Compounds and methods for modulating SMN2

Abstract

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

Description

[0001] Sequence Listing

[0002] This application is submitted with a sequence listing in electronic format. The sequence listing is provided in the form of a file titled BIOL0367WOSEQ_ST25.txt created on February 26, 2021, and is 44KB in size. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety. Technical Field

[0003] Compounds, methods and pharmaceutical compositions for regulating SMN2 RNA in cells or subjects are provided. Such compounds, methods and pharmaceutical compositions can be used to improve at least one symptom of a neurodegenerative disorder. Such symptoms include: decreased muscle strength; inability or reduced ability to sit upright, stand and / or walk; decreased neuromuscular activity; decreased electrical activity in one or more muscles; decreased respiration; inability or reduced ability to eat, drink and / or breathe without assistance; weight loss or decreased weight gain; and / or decreased survival. Background Art

[0004] Proximal spinal muscular atrophy (SMA) is a hereditary neurodegenerative disorder characterized by loss of spinal motor neurons. SMA is an early-onset autosomal recessive disease and is the main genetic cause of infant death. The severity of SMA varies from patient to patient and is therefore divided into four types. Type I SMA is the most serious form, which occurs at birth or within 6 months, and usually causes death within 2 years. Type I SMA children cannot sit or walk. Type II SMA is an intermediate form, and patients can sit, but cannot stand or walk. Type III SMA (a chronic form of the disease) patients usually develop SMA after 18 months of age (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536). Type IV SMA is a milder form and typically develops after age 18, sometimes after age 10; patients with Type IV SMA experience limited, mild motor impairments, are able to walk in adulthood and typically have no respiratory or nutritional problems (Farrar et al., Ann. Neurol., 2017, 81, 355-368; D'Amico et al., Orphanet J. of Rare Diseases, 2011, 6:71).

[0005] The molecular basis of SMA is the loss of both copies of the survival motor neuron gene 1 (SMN1), which may also be referred to as the SMN telomere and encodes a protein that is part of a multiprotein complex thought to be involved in snRNP biogenesis and recycling. An almost identical gene, SMN2 (also known as the SMN centromere), is present in a duplicated region on chromosome 5q13 and modulates disease severity. Although SMN1 and SMN2 have the potential to encode the same protein, expression of the normal SMN1 gene results only in expression of the full-length survival motor neuron (SMN) protein, whereas expression of the SMN2 gene results in two distinct protein forms, the 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 the SMN2 transcript. Thus, the predominant form of SMN2 is a truncated form 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% of full-length SMN protein and 80-90% of unstable / non-functional SMNΔ7 protein. The SMN protein plays a recognized role in the assembly of the spliceosome and may also mediate mRNA trafficking in axons and nerve terminals of neurons.

[0006] It is an object herein to provide compounds, methods and pharmaceutical compositions for the treatment of SMA. Summary of the invention

[0007] Provided herein are compounds, methods, and pharmaceutical compositions for regulating the splicing of SMN2 RNA in a cell or subject. In certain embodiments, the compound that can be used to regulate the splicing of SMN2 RNA is an oligomeric compound. In certain embodiments, the oligomeric compound increases the amount of SMN2 RNA including exon 7. In certain embodiments, the oligomeric compound increases full-length SMN2 protein expression. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide. In certain embodiments, the subject suffers from a neurodegenerative disease. In certain embodiments, the subject suffers from spinal muscular atrophy (SMA).

[0008] Also provided are methods for improving at least one symptom of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is SMA. In certain embodiments, symptoms include: decreased muscle strength; inability to sit upright, stand and / or walk or reduced ability; decreased neuromuscular activity; decreased electrical activity in one or more muscles; decreased respiration; inability to eat, drink and / or breathe without assistance or reduced ability; weight loss or reduced weight gain; and / or decreased survival. In certain embodiments, provided herein are modified oligonucleotides for treating SMA. DETAILED DESCRIPTION

[0009] It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only and are not limiting. In this article, unless otherwise specifically stated, the use of the singular includes the plural. As used herein, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "including" and other forms (such as "includes" and "included") is not limiting. Similarly, unless otherwise specifically stated, terms such as "element" or "component" cover elements and components containing one unit and elements and components containing more than one subunit.

[0010] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books and papers, and GenBank and NCBI reference sequence records are expressly incorporated by reference herein with respect to the portions of the documents discussed herein and in their entirety.

[0011] definition

[0012] Unless specific definitions are provided, the nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and drug and pharmaceutical chemistry described herein, and the procedures and techniques thereof, are those well known and commonly used in the art. Where permitted, all patents, applications, published applications, and other publications and other data cited throughout this disclosure are incorporated herein by reference in their entirety.

[0013] Unless otherwise indicated, the following terms have the following meanings:

[0014] As used herein, "2'-deoxyribonucleoside" means a nucleoside comprising a 2'-H (H) deoxyribosyl sugar moiety. In certain embodiments, the 2'-deoxyribonucleoside is a 2'-β-D-deoxyribonucleoside and comprises a 2'-β-D-deoxyribosyl sugar moiety having a β-D configuration as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxyribonucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0015] As used herein, "2'-MOE" means a 2'-OCH2CH2OCH3 group replaces the 2'-OH group of a ribosyl sugar moiety. A "2'-MOE sugar moiety" is a sugar moiety in which a 2'-OCH2CH2OCH3 group replaces 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.

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

[0017] As used herein, "2'-NMA" means a -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 having a 2'-O-CH2-C(=O)-NH-CH3 group in place of the 2'-OH group of a ribosyl sugar moiety. Unless otherwise indicated, the 2'-NMA sugar moiety is in the β-D configuration. "NMA" means ON-methylacetamide.

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

[0019] As used herein, "2'-OMe" means a 2'-OCH3 group replaces the 2'-OH group of a ribosyl sugar moiety. A "2'-OMe sugar moiety" is a sugar moiety in which a 2'-OCH3 group replaces 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.

[0020] As used herein, "2'-OMe nucleoside" means a nucleoside comprising a 2'-OMe sugar moiety. As used herein, "2'-substituted nucleoside" means a nucleoside comprising a 2'-substituted sugar moiety. As used herein, "2'-substituted" with respect to a sugar moiety means a sugar moiety comprising at least one 2'-substituent other than H or OH.

[0021] As used herein, "5-methylcytosine" means cytosine modified with a methyl group attached to position 5. 5-methylcytosine is a modified nucleobase.

[0022] As used herein, "administering" means providing a pharmaceutical agent to a subject.

[0023] As used herein, "improvement" with respect to treatment means an improvement in at least one symptom relative to the same symptom in the absence of the treatment. In certain embodiments, the improvement is a decrease in the severity or frequency of the symptom or a delayed onset of the symptom or a slowed progression of severity or frequency. In certain embodiments, the symptoms are: decreased muscle strength; inability to sit upright, stand and / or walk or reduced ability; decreased neuromuscular activity; decreased electrical activity in one or more muscles; decreased respiration; inability to eat, drink and / or breathe without assistance or reduced ability; weight loss or decreased weight gain; and / or decreased survival.

[0024] 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.

[0025] As used herein, "antisense compound" means an oligomeric compound or oligomeric duplex capable of achieving at least one antisense activity.

[0026] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

[0027] 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 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 include a furanosyl moiety.

[0028] As used herein, "cerebrospinal fluid" or "CSF" means the fluid that fills the space surrounding the brain and spinal cord. "Artificial cerebrospinal fluid" or "aCSF" means a fluid that is prepared or manufactured to have certain properties of cerebrospinal fluid.

[0029] As used herein, "cEt" means a 4' to 2' bridge replacing 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 having a 4' to 2' bridge replacing the 2'OH-group of a ribosyl sugar moiety, wherein the bridge has the formula 4'-CH(CH3)-O-2', and wherein the methyl group of the bridge is in the S configuration. "cEt" means constrained ethyl.

[0030] As used herein, "cEt nucleoside" means a nucleoside comprising a cEt sugar moiety.

[0031] As used herein, "chiral enriched population" means a plurality of molecules having the same molecular formula, wherein the number or percentage of molecules containing a particular stereochemical configuration at a particular chiral center within the population 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 when the particular chiral center is stereo random. A chiral enriched population of molecules having multiple chiral centers within each molecule may contain one or more stereo random chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound comprising a modified oligonucleotide.

[0032] As used herein, "complementarity" with respect to an oligonucleotide means that when an oligonucleotide is aligned with the core base sequence of another nucleic acid in a relative direction, at least 70% of the core bases of an oligonucleotide or one or more parts thereof can hydrogen bond to each other with the core bases of another nucleic acid or one or more parts thereof. Complementary core bases mean core bases that can form hydrogen bonds with each other. Complementary core base pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G) and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or target nucleic acids do not necessarily have core base complementarity at each nucleoside. On the contrary, some mismatches are allowed. As used herein, "complete complementarity" or "100% complementarity" with respect to an oligonucleotide or part thereof means that an oligonucleotide or part thereof is complementary to each core base of the shorter of another oligonucleotide or target nucleic acid in two oligonucleotides, or if the oligonucleotides are of the same length, then complementary at each nucleoside.

[0033] As used herein, "consecutive" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside bonds that are immediately adjacent to each other. For example, "consecutive nucleobases" means nucleobases that are immediately adjacent to each other in a sequence.

[0034] 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 hybridization mechanism involves hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.

[0035] As used herein, "internucleoside linkage" means a covalent linkage between consecutive nucleosides in an oligonucleotide. As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage. A "phosphorothioate internucleoside linkage" is a modified internucleoside linkage in which one of the non-bridging oxygen atoms of the phosphodiester internucleoside linkage is replaced by a sulfur atom.

[0036] As used herein, "mismatch" or "non-complementary" means that a nucleobase of the first oligonucleotide is not complementary to a corresponding nucleobase of the second oligonucleotide or target nucleic acid when the first oligonucleotide and the second oligonucleotide are aligned.

[0037] As used herein, "motif" means the pattern of unmodified and / or modified sugar moieties, nucleobases and / or internucleoside linkages in an oligonucleotide.

[0038] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety comprising modifications (eg, substituents) that do not form a bridge between two atoms of the sugar to form a second ring.

[0039] 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 that can pair with at least one unmodified nucleobase. "5-methylcytosine" is a modified nucleobase. A universal base is a modified nucleobase that can pair with any of the five unmodified nucleobases. As used herein, a "nucleobase sequence" means the order of consecutive nucleobases in a target nucleic acid or oligonucleotide, independent of any sugar or internucleoside linkage modification.

[0040] As used herein, "nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and 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. "Connected nucleosides" are nucleosides connected in a continuous sequence (i.e., there are no additional nucleosides between the connected nucleosides).

[0041] As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more additional features, such as a conjugated group or a terminal group. An oligomeric compound may be paired with a second oligomeric compound complementary to the first oligomeric compound or may be unpaired. A "single-stranded oligomeric compound" is an unpaired oligomeric compound. The term "oligoduplex" means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a "duplex oligomeric compound".

[0042] As used herein, "oligonucleotide" means a chain of connected nucleosides connected via an internucleoside bond, wherein each nucleoside and internucleoside bond may be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 8-50 connected nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide in which at least one nucleoside or internucleoside bond is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not include any nucleoside modification or internucleoside modification.

[0043] As used herein, "pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include an oligomeric compound and a sterile aqueous solution.

[0044] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to a subject. Certain such carriers enable the pharmaceutical composition to be formulated into, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffered solution, or sterile artificial cerebrospinal fluid.

[0045] As used herein, "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0046] Unless otherwise specified, as used herein, "RNA" means RNA transcripts and includes pre-mRNA and mature mRNA.

[0047] As used herein, "stereo-random chiral center" means a chiral center with a random stereochemical configuration in the context of a population of molecules having the same molecular formula. For example, in a population of molecules comprising a stereo-random chiral center, the number of molecules having a stereo-random chiral center with an (S) configuration may, but may not, be the same as the number of molecules having a stereo-random chiral center with an (R) configuration. The stereochemical configuration of a chiral center is considered to be random when the synthetic method is not designed to control the stereochemical configuration. In certain embodiments, the stereo-random chiral center is a stereo-random phosphorothioate internucleoside linkage.

[0048] As used herein, "subject" means a human or non-human animal.

[0049] As used herein, "sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" means a 2'-OH(H)β-D ribosyl moiety as found in RNA ("unmodified RNA sugar moiety") or a 2'-H(H)β-D deoxyribosyl moiety as found in DNA ("unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen at each of the 1', 3' and 4' positions, an oxygen at the 3' position and two hydrogens at the 5' position. As used herein, "modified sugar moiety" or "modified sugar" means a modified furanosyl sugar moiety or sugar surrogate.

[0050] As used herein, "sugar surrogate" means a modified sugar moiety other than a furanosyl moiety that can link a nucleobase to another group in an oligonucleotide (e.g., an internucleoside linkage, a conjugate group, or a terminal group). Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to a complementary oligomeric compound or a target nucleic acid.

[0051] As used herein, "standard in vivo assay" means the assay described in Example 2 and reasonable variations thereof.

[0052] As used herein, "symptom" means any physical characteristic or test result that indicates the presence or extent of a disease or condition. In certain embodiments, the symptom is obvious to the subject or to a medical professional examining or testing the subject.

[0053] As used herein, "target nucleic acid" means the nucleic acid that an antisense compound is designed to affect.

[0054] As used herein, "target region" means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

[0055] As used herein, "terminal group" means a chemical group or atom group covalently attached to the terminus of an oligonucleotide.

[0056] As used herein, "therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to a subject. For example, a therapeutically effective amount improves the symptoms of a disease.

[0057] Certain embodiments

[0058] The present disclosure provides the following non-limiting numbered embodiments:

[0059] Embodiment 1. An oligomeric compound comprising a modified oligonucleotide consisting of 16, 17, 18, 19 or 20 linked nucleosides and a nucleobase sequence comprising at least 15 or at least 16 consecutive nucleobases of any one of the nucleobase sequences 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.

[0060] Embodiment 2. An oligomeric compound comprising a modified oligonucleotide consisting of 17, 18, 19 or 20 linked nucleosides and a nucleobase sequence comprising at least 15, at least 16 or at least 17 consecutive nucleobases of any one of the nucleobase sequences SEQ ID NO: 20-27, 29-30 or 32-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0061] Embodiment 3. An oligomeric compound comprising a modified oligonucleotide consisting of 18, 19 or 20 linked nucleosides and a nucleobase sequence comprising at least 15, at least 16, or at least 17 or at least 18 consecutive nucleobases of any one of the nucleobase sequences SEQ ID NO: 20-27, 30 or 33-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0062] Embodiment 4. An oligomeric compound comprising a modified oligonucleotide consisting of 19 or 20 linked nucleosides and a nucleobase sequence comprising at least 15, at least 16, or at least 17, at least 18 or at least 19 consecutive nucleobases of any one of the nucleobase sequences SEQ ID NO: 20, 22, 24-27, 30, 33-50, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0063] Embodiment 5. An oligomeric compound comprising a modified oligonucleotide consisting of 20 linked nucleosides and a nucleobase sequence comprising at least 15, at least 16, or at least 17, at least 18, at least 19 or at least 20 consecutive nucleobases of any one of the nucleobase sequences SEQ ID NO: 20, 22, 25, 27, 35, 39-46 or 49, wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.

[0064] Embodiment 6. An oligomeric compound as described in any of embodiments 1-5, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, 85%, 87.5%, 88.2%, 89%, 89.4%, 90%, 93.7%, 94%, 94.7%, 95% or 100% complementary to the nucleobase sequence of SEQ ID NO:1 when measured over the entire nucleobase sequence of the modified oligonucleotide.

[0065] Embodiment 7. An oligomeric compound according to any one of Embodiments 1-6, wherein the modified oligonucleotide has an internucleoside linkage motif (5' to 3') selected from the following: sosososssssssssssss, ssosssssssssssoss, ssosssssosssssoss, ssosssosssosssoss, soossssssssssooss, sooosssssssssooss, sooossssssssoooss, ssssssssooosssssss, ssossssssssssssss, sssssossssssssssss, sssssssossssssssss, sssssssssossssssss, sssssssssssossssss, sssssssssssssossss, sssssssssssssssoss, sossssssssssssoss, sosssssssssosssss, sosssssssosssssss, sosssssosssssssss, sosssosssssssssss, sssssosssssssssoss, sssssssosssssssoss, sssssssssosssssoss, sssssssssssosssoss, sssssssssssssososs, soossssssssssssss, sssoossssssssssss, ssssssoossssssssss, ssssssssoossssssss, ssssssssssoossssss, ssssssssssssoossss, ssssssssssssssooss, ssssssssoooossssss, ssoooosssssssssss, ssssoooosssssssss, sssssssssoooosssss, sssssssssssoooosss, ssssssssssssooooss, sssssssooooossssss, sssssssoooooosssss, sooosssssssoooss, ssssssooooooosssss, sssssssssssssssoss, sssssssssssssosss, ssssssssssssssooss, sssssssssssssososs, ssssssssssssosssss, ssssssssssssososss, sssssssssssossosss, ssssssssssosssssss, ssssssssssosssosss, sssssssssosssssoss,ssssssssosssss、ssssssssssss、ssssssssss、ssssssssoosssssssss、s ...、sosssssssssssssss、sosssssssssssss ss、soossssssssssss、osssssssssssssso、s ...、sssssssssssssssssss、sssssssssssssssssss、ssssssssssssssssssss、sss sssssssssss、sssssssssssss、ssssssssssss、ssssssssssss、sosssssssssssssssss、sossssssssssssssssssss、sosssssssssssssssssss、sosssssssssssssssss、sosssssssssssssssss、sossssssssssssssss、sosssssssssssssss、sosss sssssssssss、soosssssssssssss、ssssssssssssssss、sssssssssssssssss、ssssssssssssssss、osssssssssssssssssss、sssssssssssssss、sssssssssssssss、sssssssssssssss、ssssssssssssssss、ssssssssssssssss、sssssssssssssssssss、sssssssssssssssss ssssssssssssoss、ssssssssssssss、ssssssssssssssss、sssssssssssssssss、soss ...、sosssssssssssssssssss、sossssssssssssssssss、sosssssssssssssssss sossssssssssss、sssssssssooooss、ssssssssssss、ssssssssssss、ssssssssssss、sssssssssooossssssss、s ...ooooossssss、sssssssssss、ssssssssss ooooooossss、sssssssssss、ssossssssssss、ssosssssssssss、ssossssssssssss、ssossssssssssss、ssosssssssssss、ssossssssssss、ssoooosssssssssss、sooosssssssssssss、sooossssssssssssss、sooossssssssssssss、ssssssssssssssss、ssooos ...、sssssssssssssssss、sssssssssssssssss、sssssssssssssssss、ssssssoooosssss, ssssssooooosssss, sssssoooooosssss, ssssoooosssssss, ssssooooooossss, sssosssosssosss, ssosssssssssss, ssossossossssss, ssossossossosss, ssossossososs, ssoooosssssssss, soosssssssssooss, soooosssssssoooss, and soooossssssoooss; wherein 's' represents a phosphorothioate internucleoside linkage and 'o' represents a phosphodiester internucleoside linkage.

[0066] Embodiment 8. An oligomeric compound as described in any of embodiments 1-6, wherein the modified oligonucleotide has an internucleoside linkage motif selected from the following: sssssssssssssssxs and ssssssssssssssssx; wherein 's' represents a phosphorothioate internucleoside linkage, 'o' represents a phosphodiester internucleoside linkage and "x" represents a methoxypropylphosphonate internucleoside linkage.

[0067] Embodiment 9. An oligomeric compound as described in any of embodiments 1-6, wherein the modified oligonucleotide has an internucleoside linkage motif selected from the following: zzzzzzzzzzzzzzzzzzz, sssssssssssszzzz, ssssszzzzzzssssss, zzooooooooooooozz, zzzzooooooooooozz, zzzzzzooooooooozz, zzzzzzzzooooooooozz and ssoooooooooooooss; wherein 's' represents phosphorothioate internucleoside linkages, 'o' represents phosphodiester internucleoside linkages and "z" represents methylsulfonyl phosphoramidate internucleoside linkages.

[0068] Embodiment 10. The oligomeric compound according to any one of Embodiments 1-9, wherein the modified oligonucleotide has a glycosyl motif (5' to 3') selected from the following: eeeeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeeee, eeeeeeeeeeeeeeeee, nnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnnn, nnnnnnnnnnnnnnnnnnnnn, nennnnneneennnnnnn, nnnnnnnnnnnenneen, nennnnneneenenneen, nnnnnnnnnnnnnnnnnnne, nnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnny, nnnnnnnnnnnnnnnnnnndd, nnnnnnnnnnnnnnnnnnned, nnnnnnnnnnnnnnnnnnnde, nnnnnnnnnnnnnnnnnnnee, eeeeeeeeeeeeeeeeeeedd, eeeeeeeeeeeeeeeeeeeed, eeeeeeeeeeeeeeeeeeede, nnnnnnnnnnnnnnnnnnnd, nnnnnnnnnnnnnnnnnnne, eeeeeeeeeeeeeeeeeeed, keekeekeekeekeeeek, keeeekeeekeeekeeeek, keeeeekeeeeekeeeek, keeeeeeekeeeeeeeek, keeeeeeeeeeeeeeeek, eeekeekeekeekeekek, eeekeekeekeekeekee, eeeeeeekeekeekeekee, eeeeeeekeekeekeeeee, eeeeeeekeeeeekeeeee, keekeekeekeeeeeeee, eeeeeeeeekeekeekeek, keekeekeeeeeeeeeee, eeeeeeeeeeeekeekeek, keekeeeeeeeeeeeeee, eeeeeeeeeeeeeeekeek, keekeekeekeekeeek, keeeekeeekeeekeeek, keeeekeeeeekeeeek, keeeeeeekeeeeeeek, keeeeeeeeeeeeeeek, eekeekeekeekeekek,eekeekeekeekeekee, eeeeekeekeekeekee, eeeeekeekeekeeeee, eeeeekeeeeeekeeeee, keekeekeekeeeeeee, eeeeeeekeekeekeek, keekeekeeeeeeeeee, eeeeeeeeekeekeek, keekeeee eeeeeeee, eeeeeeeeeeeeekeek, keekeekeekeekeek, keeekeekeekeekeek, keeeekeeeekeeeek, keeeeeeekeeeeeek, keeeeeeeeeeeeek, kekeekeekeekeeke, eekeekeekeekeeke, eeeeekee keekeeke, eeeeekeekeekeeee, eeeeekeeeeekeeee, keekeekeekeeeeee, eeeeeekeekeeek, keekeeeeeeeeeeee, eeeeeeeeeeeeeekeek, eeeeeeeeeeeeeeeeeeeed, eeeeeeeeeeeeeeeeeeey, ennnnnnnnnnnnnnnnnnn, and ennnnnnnnnnnnnnnnnnnne; wherein 'e' represents a 2'-MOE sugar moiety, 'n' represents a 2'-NMA sugar moiety, 'k' represents a cEt sugar moiety, 'd' represents a 2'-β-D-deoxyribosyl sugar moiety, and 'y' represents a 2'-OMe sugar moiety.

[0069] Embodiment 11. An oligomeric compound as described in any of embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3') selected from the following: nnnnnnnnnnnnnnenn and nnnnnnnnnnnnnnnnenen, wherein 'e' represents a 2'-MOE sugar moiety and 'n' represents a 2'-NMA sugar moiety.

[0070] Embodiment 12. An oligomeric compound as described in any one of embodiments 1-9, wherein the modified oligonucleotide has a sugar motif (5' to 3')qqnqqqqqnqnnqnqqnn, wherein each 'n' represents a 2'-NMA sugar moiety and each 'q' is independently selected from a 2'-O-(N,N-dimethyl)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.

[0071] Embodiment 13. The oligomeric compound of any one of embodiments 1-9, wherein the modified oligonucleotide comprises at least one modified sugar moiety.

[0072] Embodiment 14. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one bicyclic sugar moiety.

[0073] 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.

[0074] Embodiment 16. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one non-bicyclic modified sugar moiety.

[0075] 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.

[0076] Embodiment 18. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one sugar replacement.

[0077] Embodiment 19. The oligomeric compound of embodiment 18, wherein the sugar surrogate is any one of morpholino, modified morpholino, PNA, THP and F-HNA.

[0078] 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.

[0079] Embodiment 21. The oligomeric compound of embodiment 20, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

[0080] Embodiment 22. The oligomeric compound of embodiment 20 or embodiment 21, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0081] 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.

[0082] Embodiment 24. The oligomeric compound of embodiment 20, 22 or 23, wherein each internucleoside linkage is independently selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

[0083] Embodiment 25. An oligomeric compound as described in any one of embodiments 13-19, wherein the modified oligonucleotide has an internucleoside linkage motif (5' to 3') selected from the following: sososssssssssssss, soossssssssssssss, sosssosssssssssss, sosssssossssssssss, sossssssosssssssss, sossssssssossssssss, sssoosssssssss, sssssssssoosssssss and sssssssssssoossssss; wherein 's' represents phosphorothioate internucleoside linkages and 'o' represents phosphodiester internucleoside linkages.

[0084] Embodiment 26. The oligomeric compound of any one of embodiments 1-25, wherein the modified oligonucleotide comprises a modified nucleobase.

[0085] Embodiment 27. The oligomeric compound of embodiment 26, wherein the modified nucleobase is 5-methylcytosine.

[0086] 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.

[0087] Embodiment 29. The oligomeric compound of any one of embodiments 1-28, wherein the modified oligonucleotide comprises 1 or 2 non-complementary nucleobases.

[0088] Embodiment 30. The oligomeric compound of any one of embodiments 1-29, wherein the modified oligonucleotide comprises 1 or 2 cleavable moieties.

[0089] Embodiment 31. The oligomeric compound of embodiment 30, wherein the cleavable moiety is a phosphodiester internucleoside linkage.

[0090] Embodiment 32. The oligomeric compound of any one of embodiments 1-31, which consists of the modified oligonucleotide.

[0091] Embodiment 33. The oligomeric compound of any one of embodiments 1-32, wherein the oligomeric compound is a single-stranded oligomeric compound.

[0092] Embodiment 34. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: m C es A eo m C es T eo T es T es m C es A es T es A es A es T es G es m C es T es G es G es m C e (SEQ ID NO: 21), wherein:

[0093] A = adenine nucleobase,

[0094] m C=5-methylcytosine nucleobase,

[0095] G = guanine nucleobase,

[0096] T = thymine nucleobase,

[0097] e=2'-MOE sugar moiety,

[0098] s = phosphorothioate internucleoside linkage, and

[0099] o = phosphodiester internucleoside linkage.

[0100] Embodiment 35. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: eo T es m C es A es m C es T es T es T es m C es A es T es A es A es T es G esm C es T es G es G eo m C e (SEQ ID NO:22), wherein:

[0101] A = adenine nucleobase,

[0102] m C=5-methylcytosine nucleobase,

[0103] G = guanine nucleobase,

[0104] T = thymine nucleobase,

[0105] e=2'-MOE sugar moiety,

[0106] s = phosphorothioate internucleoside linkage, and

[0107] o = phosphodiester internucleoside linkage.

[0108] Embodiment 36. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: eo T ns m C ns A ns m C ns T ns T ns T ns m C ns A ns T ns A ns A ns T ns G ns m C ns T ns G ns G no m C e (SEQ ID NO:22), wherein:

[0109] A = adenine nucleobase,

[0110] m C=5-methylcytosine nucleobase,

[0111] G = guanine nucleobase,

[0112] T = thymine nucleobase,

[0113] e=2'-MOE sugar moiety,

[0114] n=2'-NMA sugar moiety,

[0115] s = phosphorothioate internucleoside linkage, and

[0116] o = phosphodiester internucleoside linkage.

[0117] Embodiment 37. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: m C ns A no m C ns T no T ns T ns m C ns A ns T ns A ns A ns T ns G ns m C ns T ns G ns G ns m C n (SEQ ID NO: 21), wherein:

[0118] A = adenine nucleobase,

[0119] m C=5-methylcytosine nucleobase,

[0120] G = guanine nucleobase,

[0121] T = thymine nucleobase,

[0122] n=2'-NMA sugar moiety,

[0123] s = phosphorothioate internucleoside linkage, and

[0124] o = phosphodiester internucleoside linkage.

[0125] Embodiment 38. A modified oligonucleotide according to the following chemical structure:

[0126]

[0127] (SEQ ID NO: 21), or a salt thereof.

[0128] Embodiment 39. The modified oligonucleotide of embodiment 38, which is a sodium salt or a potassium salt.

[0129] Embodiment 40. A modified oligonucleotide according to the following chemical structure:

[0130]

[0131] (SEQ ID NO:21).

[0132] Embodiment 41. A modified oligonucleotide according to the following chemical structure:

[0133]

[0134] (SEQ ID NO: 22), or a salt thereof.

[0135] Embodiment 42. The modified oligonucleotide of embodiment 41, which is a sodium salt or a potassium salt.

[0136] Embodiment 43. A modified oligonucleotide according to the following chemical structure:

[0137]

[0138] (SEQ ID NO:22).

[0139] Embodiment 44. A modified oligonucleotide according to the following chemical structure:

[0140]

[0141] (SEQ ID NO: 22), or a salt thereof.

[0142] Embodiment 45. The modified oligonucleotide of embodiment 44, which is a sodium salt or a potassium salt.

[0143] Embodiment 46. A modified oligonucleotide corresponding to the following chemical structure:

[0144]

[0145] (SEQ ID NO:22).

[0146] Embodiment 47. A modified oligonucleotide according to the following chemical structure:

[0147]

[0148] (SEQ ID NO: 21), or a salt thereof.

[0149] Embodiment 48. The modified oligonucleotide of embodiment 47, which is a sodium salt or a potassium salt.

[0150] Embodiment 49. A modified oligonucleotide according to the following chemical structure:

[0151]

[0152] (SEQ ID NO:21).

[0153] 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.

[0154] 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.

[0155] Embodiment 52. The pharmaceutical composition of Embodiment 51, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and artificial CSF (aCSF).

[0156] Embodiment 53. The pharmaceutical composition of Embodiment 51, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and PBS.

[0157] Embodiment 54. A chirally enriched population of modified oligonucleotides as described in any of Embodiments 38-49, wherein the population is enriched for at least one modified oligonucleotide having a specific phosphorothioate internucleoside linkage with a specific stereochemical configuration.

[0158] Embodiment 55. The chirally enriched population of Embodiment 54, wherein the population is enriched for at least one modified oligonucleotide having a specific phosphorothioate internucleoside linkage of (Sp) configuration.

[0159] Embodiment 56. The chirally enriched population of Embodiment 54, wherein the population is enriched for at least one modified oligonucleotide having a specific phosphorothioate internucleoside linkage of (Rp) configuration.

[0160] Embodiment 57. The chirally enriched population of Embodiment 54, wherein the population is enriched for modified oligonucleotides having a specific independently selected stereochemical configuration at each phosphorothioate internucleoside linkage.

[0161] Embodiment 58. A chiral enriched population as described in Embodiment 57, wherein the population is enriched for modified oligonucleotides having a (Sp) configuration at each phosphorothioate internucleoside linkage or modified oligonucleotides having a (Rp) configuration at each phosphorothioate internucleoside linkage.

[0162] Embodiment 59. A chirally enriched population as described in Embodiment 57, wherein the population is enriched for modified oligonucleotides having the (Rp) configuration at one particular phosphorothioate internucleoside linkage and the (Sp) configuration at each of the remaining phosphorothioate internucleoside linkages.

[0163] Embodiment 60. The chirally enriched population of Embodiment 57, wherein the population is enriched for modified oligonucleotides having at least 3 consecutive phosphorothioate internucleoside linkages in the configurations of Sp, Sp, and Rp in the 5' to 3' direction.

[0164] Embodiment 61. A population of modified oligonucleotides as described in any one of embodiments 38-49, wherein all phosphorothioate internucleoside linkages of the modified oligonucleotides are stereorandom.

[0165] Embodiment 62. A method of treating a disease associated with SMN1 or SMN2, the method comprising administering to a subject having or at risk of developing a disease associated with SMN1 or SMN2 a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 50-53; and thereby treating the disease associated with SMN1 or SMN2.

[0166] Embodiment 63. The method of embodiment 62, wherein the disease associated with SMN1 or SMN2 is a neurodegenerative disease.

[0167] Embodiment 64. The method of embodiment 63, wherein the neurodegenerative disease is spinal muscular atrophy (SMA).

[0168] Embodiment 65. The method of embodiment 64, wherein the SMA is any one of type I SMA, type II SMA, type III SMA, or type IV SMA.

[0169] Embodiment 66. The method of embodiment 64 or embodiment 65, wherein at least one symptom of SMA is improved.

[0170] Embodiment 67. A method as described in embodiment 66, wherein the symptom is any of the following: decreased muscle strength; inability or reduced ability to sit upright, stand and / or walk; decreased neuromuscular activity; decreased electrical activity in one or more muscles; decreased respiration; inability or reduced ability to eat, drink and / or breathe without assistance; weight loss or decreased weight gain; and / or decreased survival rate.

[0171] Embodiment 68. The method of any one of embodiments 62-67, wherein the pharmaceutical composition is administered to the central nervous system or systemically.

[0172] Embodiment 69. The method of embodiment 68, wherein the pharmaceutical composition is administered to the central nervous system and systemically.

[0173] Embodiment 70. The method of any one of embodiments 62-67, wherein the pharmaceutical composition is administered any one of intrathecally, systemically, subcutaneously, or intramuscularly.

[0174] Embodiment 71. A method of increasing SMN2 RNA comprising exon 7, the method comprising contacting a cell, tissue or organ with an oligomeric compound of any one of embodiments 1-37, a modified oligonucleotide of any one of embodiments 38-49, or a pharmaceutical composition of any one of embodiments 50-53.

[0175] Certain oligonucleotides

[0176] In certain embodiments, provided herein are oligomeric compounds comprising an oligonucleotide consisting of connected nucleosides. The oligonucleotide may be an unmodified oligonucleotide (RNA or DNA) or may be a modified oligonucleotide. Relative to unmodified RNA or DNA, the modified oligonucleotide comprises at least one modification. That is, the modified oligonucleotide comprises at least one modified nucleoside (comprising a modified sugar moiety and / or a modified core base) and / or at least one modified internucleoside linkage.

[0177] Certain modified nucleosides

[0178] Modified nucleosides comprise a modified sugar moiety, or a modified nucleobase, or both a modified sugar moiety and a modified nucleobase.

[0179] Certain sugar moieties

[0180] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogate may contain one or more substitutions corresponding to other types of modified sugar moieties.

[0181] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety comprising a furanosyl ring having one or more substituents, none of which bridge the two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents may be located at any position of the furanosyl group, including but not limited to substituents at the 2', 4' and / or 5' positions. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moiety are branched. Examples of 2'-substituents suitable 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-alkylacetamides, for example, 2'-ON-methylacetamide ("NMA"), 2'-ON-dimethylacetamide, 2'-ON-ethylacetamide or 2'-ON-propylacetamide. For example, see US 6,147,200, Prakash et al., 2003, Org. Lett., 5, 403-6. "2'-ON-methylacetamide nucleoside" or "2'-NMA nucleoside" is shown below:

[0182]

[0183] In certain embodiments, the 2'-substituent is selected from the group consisting of halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C 10 Alkoxy, O-C1-C 10 Substituted alkoxy, O-C1-C 10 Alkyl, O-C1-C 10 Substituted alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ) or OCH2C(=O)-N(R m )(R n ), where each R m and Rn are independently H, an amino protecting group or a substituted or unsubstituted C1-C 10 alkyl, and 2'-substituents described in Cook et al., US6,531,584; Cook et al., US5,859,221; and Cook et al., US6,005,087. Certain embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from the following: hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of 4'-substituents suitable 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 5'-substituents suitable for non-bicyclic modified sugar moieties include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugar moiety comprises more than one non-bridging sugar substituent, such as 2'-F-5'-methyl sugar moiety and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008 / 101157 and Rajeev et al., US 2013 / 0203836.

[0184] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), O(CH2), ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2 and N-substituted acetamides (OCH2C(=O)-N(R m )(R n )), where each R m and R n are independently H, an amino protecting group or a substituted or unsubstituted C1-C 10 Alkyl, for example, OCH2C(=O)-N(H)CH3 ("NMA").

[0185] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from: F, OCF 3、OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 (“NMA”).

[0186] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of: F, OCH3, OCH2CH2OCH3, and OCH2C(=O)-N(H)CH3.

[0187] Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, thereby generating a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' bridged sugar substituents include, but are not limited to, 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE"), and the like (see, e.g., Seth et al., US Pat. No. 7,399,845; Bhat et al., US Pat. No. 7,569,686; Swayze et al., US Pat. No. 7,741,457 and Swayze et al., US Pat. No. 7,741,457). 8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,426), 4'-C(R a R b )-N(R)-O-2'、4'-C(R a R b)-ON(R)-2', 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2', wherein each R, R a and R b are independently H, a protecting group or a C1-C 12 Alkyl (see, for example, Imanishi et al., US 7,427,672).

[0188] In certain embodiments, such 4' to 2' bridges independently comprise 1 to 4 linked groups independently selected from: -[C(R a )(R b )] n -、-[C(R a )(R b )] n -O-、-C(R a )=C(R b )-、-C(R a )=N-、-C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-、-S(=O) x -and-N(R a )-;

[0189] in:

[0190] x is 0, 1, or 2;

[0191] n is 1, 2, 3 or 4;

[0192] Each R a and R b are independently H, a protecting group, a hydroxyl group, a C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxyl (S(=O)-J1); and

[0193] Each J1 and J2 is independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1-C 12 Aminoalkyl or a protecting group.

[0194] Other bicyclic sugar moieties are known in the art, see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 836 2-8379; Wengel et al., US7,053,207; Imanishi et al., US6,268,490; Imanishi et al., US6,770,748; Imanishi et al., USRE44,779; Wengel et al., US6,794,499; Wengel et al., US6,670,461; Wengel et al., US7,034,133; Wengel et al., US8,080,644; Wengel et al., US8,034,909; Wengel et al., US8,153,365; Wengel et al., US7,572,582; and Ramasamy et al., US6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO 1999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., US7,547,684; Seth et al., US7,666,854; Seth et al., US8,088,746; Seth et al., US7,750,131; Seth et al., US8,030,467; Seth et al., US8,268,980; Seth et al., US8,546,556; Seth et al., US8,530,640; Migawa et al., US9,012,421; Seth et al., US8,501,805; and Allerson et al., U.S. Patent Publication No. US2008 / 0039618 and Migawa et al., U.S. Patent Publication No. US2015 / 0191727.

[0195] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, LNA nucleosides (described herein) may be in the α-L configuration or the β-D configuration.

[0196]

[0197] α-L-methyleneoxy (4'-CH2-O-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides and have shown antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this article, the general description of bicyclic nucleosides includes two isomeric configurations. Unless otherwise specified, when the position of a specific bicyclic nucleoside (e.g., LNA or cEt) is identified in the embodiments illustrated in this article, it is in the β-D configuration.

[0198] In certain embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (eg, 5'-substituted and 4'-2' bridged sugars).

[0199] In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain such embodiments, the oxygen atom of the sugar moiety is replaced by, for example, a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described herein. For example, some sugar surrogates include 4'-sulfur atoms and substitutions at the 2' position (see, for example, Bhat et al., US7,875,733 and Bhat et al., US7,939,677) and / or 5' positions.

[0200] In certain embodiments, the sugar surrogate comprises a ring having other than 5 atoms. For example, in certain embodiments, the sugar surrogate 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 acids ("HNA"), anitol nucleic acids ("ANA"), mannitol nucleic acids ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:

[0201]

[0202] ("F-HNA", see, e.g., Swayze et al., US8,088,904; Swayze et al., US8,440,803; Swayze et al., US8,796,437; and Swayze et al., US9,005,906; F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran) and nucleosides comprising other modified THP compounds having the formula:

[0203]

[0204] Wherein, independently, for each of the modified THP nucleosides:

[0205] Bx is the nucleobase part;

[0206] T3 and T4 are each independently an internucleoside linking group that links the modified THP nucleoside to the remainder of the oligonucleotide, or one of T3 and T4 is an internucleoside linking group that links the modified THP nucleoside to the remainder of the oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugating group, or a 5' or 3'-terminal group;

[0207] q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and

[0208] R1 and R2 are each independently selected from: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein X is O, S or NJ1, and J1, J2 and J3 are each independently H or C1-C6 alkyl.

[0209] In certain embodiments, a modified THP nucleoside is provided wherein each of q1, q2, q3, q4, q5, q6 and q7 is H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is not H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, a modified THP nucleoside is provided wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.

[0210] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholinyl 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., US5,698,685; Summerton et al., US5,166,315; Summerton et al., US5,185,444; and Summerton et al., US5,034,506). As used herein, the term "morpholinyl" means a sugar surrogate having the following structure:

[0211]

[0212] In certain embodiments, the morpholinyl group can be modified, for example, by adding or changing various substituents compared to the above-described morpholinyl structure. Such sugar surrogates are referred to herein as "modified morpholinyl groups".

[0213] In certain embodiments, the sugar surrogate comprises a non-cyclic moiety. Examples of nucleosides and oligonucleotides comprising such non-cyclic sugar surrogates include, but are not limited to, peptide nucleic acids ("PNAs"), non-cyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865) and Manoharan et al., nucleosides and oligonucleotides described in WO2011 / 133876.

[0214] Numerous other bicyclic and tricyclic sugars and sugar surrogate ring systems are known in the art for use in modified nucleosides.

[0215] Certain modified nucleobases

[0216] In certain embodiments, the modified oligonucleotide comprises one or more nucleosides comprising unmodified nucleobases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides comprising modified nucleobases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides not comprising nucleobases, referred to as abasic nucleosides.

[0217] In certain embodiments, the modified nucleobase is selected from the group consisting of: a 5-substituted pyrimidine, a 6-azapyrimidine, an alkyl or alkynyl substituted pyrimidine, an alkyl substituted purine, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobase is selected from the group consisting of: 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy, 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-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, hybrid bases, size-expanded bases and fluorinated bases. Other modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenthiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone.Other nucleobases include those disclosed by Merigan et al., US 3,687,808; The Concise Encyclopedia Of Polymer Science And Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, edited by Crooke, ST and Lebleu, B., CRC Press, 1993, 273-288; and Chapters 6 and 15, Antisense Drug Technology, edited by Crooke ST, CRC Press, 2008, 163-166 and 442-443.

[0218] Disclosures that teach the preparation of some of the modified nucleobases described above, as well as other modified nucleobases, include, but are not limited to: Manoharan et al., US 2003 / 0158403; Manoharan et al., US 2003 / 0175906; Dinh et al., US 4,845,205; Spielvogel et al., US 5,130,302; Rogers et al., US 5,134,066; Bischofberger et al., US 5 ,175,273; Urdea et al., US5,367,066; Benner et al., US5,432,272; Matteucci et al., US5,434,257; Gmeiner et al., US5,457,187; Cook et al., US5,459,255; Froehler et al., US5,484,908; Matteucci et al., US5,502,177; Hawkins et al., US5,525, 711; Haralambidis et al., US5,552,540; Cook et al., US5,587,469; Froehler et al., US5,594,121; Switzer et al., US5,596,091; Cook et al., US5,614,617; Froehler et al., US5,645,985; Cook et al., US5,681,941; Cook et al., US5,811,534; Cook et al. Cook et al., US5,750,692; Cook et al., US5,948,903; Cook et al., US5,587,470; Cook et al., US5,457,191; Matteucci et al., US5,763,588; Froehler et al., US5,830,653; Cook et al., US5,808,027; Cook et al., US6,166,199; and Matteucci et al., US6,005,096.

[0219] Certain modified internucleoside linkages

[0220] In certain embodiments, the nucleosides of the modified oligonucleotides can be linked together using any internucleoside linkage. Two main categories of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to: phosphodiester, which contains a phosphodiester bond (P(O2)=O) (also referred to as unmodified or naturally occurring linkages); phosphotriester; methylphosphonate; methoxypropylphosphonate ("MOP"); phosphoramidate; methylsulfonylphosphoramidate; thiophosphate (P(O2)=S) and dithiophosphate (HS-P=S). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to: methylenemethylimino (-CH2-N(CH3)-O-CH2-); thiodiester; thiocarbonylcarbamate (-OC(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N, N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to naturally occurring phosphate linkages, modified internucleoside linkages can be used to alter (usually increase) the nuclease resistance of oligonucleotides. In certain embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or separated enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0221] Representative internucleoside linkages with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. The modified oligonucleotides comprising internucleoside linkages with chiral centers can be prepared into a population of modified oligonucleotides comprising stereo-irregular internucleoside linkages, or a population of modified oligonucleotides comprising thiophosphate internucleoside linkages in a specific stereochemical configuration. In certain embodiments, the population of modified oligonucleotides comprises a thiophosphate internucleoside linkage in which all thiophosphate internucleoside linkages are stereo-irregular. Such modified oligonucleotides can be generated using a synthetic method that allows the stereochemical configuration of each thiophosphate internucleoside linkage to be randomly selected. Nevertheless, as those skilled in the art fully understand, each individual thiophosphate of each individual oligonucleotide molecule has a determined stereo configuration. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides comprising one or more specific thiophosphate internucleoside linkages in a specific independently selected stereochemical configuration. In certain embodiments, a specific phosphorothioate nucleoside linkage of a specific configuration is present in at least 65% of the molecules in the population. In certain embodiments, a specific phosphorothioate nucleoside linkage of a specific configuration is present in at least 70% of the molecules in the population. In certain embodiments, a specific phosphorothioate nucleoside linkage of a specific configuration is present in at least 80% of the molecules in the population. In certain embodiments, a specific phosphorothioate nucleoside linkage of a specific configuration is present in at least 90% of the molecules in the population. In certain embodiments, a specific phosphorothioate nucleoside linkage of a specific configuration is present in at least 99% of the molecules in the population. Such chiral enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, such as Oka et al., JACS, 2003, 125, 8307; Wan et al., Nuc. Acid. Res., 2014, 42, 13456 and the methods described in WO 2017 / 015555. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides having at least one indicated thiophosphate in (Sp) configuration. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides having at least one thiophosphate in (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and / or (Sp) thiophosphates respectively comprise one or more of the following formulae, wherein "B" indicates a nucleobase:

[0222]

[0223] Unless otherwise indicated, the chiral internucleoside linkages of the modified oligonucleotides described herein can be stereorandom or in a specific stereochemical configuration.

[0224] In certain embodiments, the modified oligonucleotide comprises an internucleoside motif (5' to 3')sooosssssssssssssss. In certain embodiments, the specific stereochemical configuration of the modified oligonucleotide is (5' to 3')Sp-ooo-Sp-Sp-Sp-Rp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp or Sp-ooo-Sp-Sp-Sp-Rp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp-Sp; wherein each 'Sp' represents a phosphorothioate internucleoside linkage in the S configuration; Rp represents a phosphorothioate internucleoside linkage in the R configuration; and 'o' represents a phosphodiester internucleoside linkage.

[0225] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, 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'), methylal (3'-O-CH2-O-5'), methoxypropyl and thiomethylal (3'-S-CH2-O-5'). Other neutral internucleoside linkages include nonionic linkages comprising siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Shanghvi and P D Cook, eds., ACSSymposium Series 580; Chapters 3 and 4, 40-65). Other neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 components.

[0226] In certain embodiments, the modified internucleoside linkage is any of those described in WO 2021 / 030778, which is incorporated herein by reference.

[0227] Certain motifs

[0228] In certain embodiments, the modified oligonucleotide comprises one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises one or more modified nucleosides comprising a modified core base. In certain embodiments, the modified oligonucleotide comprises one or more modified internucleoside linkages. In such embodiments, the modified, unmodified and differently modified sugar moieties, core bases and / or internucleoside linkages of the modified oligonucleotide define patterns or motifs. In certain embodiments, the patterns of sugar moieties, core bases and internucleoside linkages are each independent of one another. Therefore, modified oligonucleotides and described by their sugar motifs, core base motifs and / or internucleoside linkage motifs (as used herein, the core base motif describes the modification of the core base that is unrelated to the sequence of the core base).

[0229] Certain sugar motifs

[0230] In certain embodiments, the oligonucleotide comprises one or more types of modified sugars and / or unmodified sugar moieties arranged in a defined pattern or sugar motif along the oligonucleotide or portion thereof. In certain cases, such sugar motifs include, but are not limited to, any sugar modification discussed herein.

[0231] In certain embodiments, the modified oligonucleotide has a gapmer motif, which is defined by two external regions or "flanks" and a central or internal region or "gap". The three regions (5'-flanks, gaps and 3'-flanks) of the gapmer motif form a continuous sequence of nucleosides, wherein at least some sugar moieties of the nucleosides of each flank are different from at least some sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides closest to the gap in each flank (the most 3' end nucleosides of the 5'-flanks and the most 5' end nucleosides of the 3'-flanks) are different from the sugar moieties of the adjacent gap nucleosides, thus defining the boundary between the flanks and the gap (i.e., flank / gap junctions). In certain embodiments, the sugar moieties in the gap are identical to each other. In certain embodiments, the gap includes one or more nucleosides, and its sugar moieties are different from the sugar moieties of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two flanks are identical to each other (symmetrical gapmers). In certain embodiments, the 5'-flanking sugar motif is different from the 3'-flanking sugar motif (asymmetric spacer).

[0232] In certain embodiments, the flank of a spacer comprises 1-6 nucleosides. In certain embodiments, each nucleoside of each flank of a spacer 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 flank of a spacer comprises a modified sugar moiety.

[0233] In certain embodiments, the gap of the spacer comprises 7-12 nucleosides. In certain embodiments, each nucleoside of the gap of the spacer comprises a 2-deoxyribosyl sugar moiety. In certain embodiments, at least one nucleoside of the gap of the spacer comprises a modified sugar moiety and each of the remaining nucleosides comprises a 2'-deoxyribosyl sugar moiety.

[0234] In this article, the length (number of nucleosides) of the three regions of the spacer can be provided using the notation [number of nucleosides in the 5'-flank]-[number of nucleosides in the gap]-[number of nucleosides in the 3'-flank]. Therefore, the 5-10-5 spacer is composed of 5 connected nucleosides in each flank and 10 connected nucleosides in the gap. In the case where this type of nomenclature is subsequently specifically modified, the modification is a modification in each sugar moiety of each flank and the gap nucleosides include a 2'-deoxyribosyl sugar moiety. Therefore, the 5-10-5MOE spacer is composed of 5 connected 2'-MOE nucleosides in the 5'-flank, 10 connected 2'-deoxyribonucleosides in the gap, and 5 connected 2'-MOE nucleosides in the 3'-flank.

[0235] In certain embodiments, each nucleoside or a portion thereof of a modified oligonucleotide comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, a sugar surrogate or a 2'-deoxyribosyl sugar moiety. In certain embodiments, the 2'-substituted sugar moiety is selected from 2'-MOE sugar moiety, 2'-NMA sugar moiety, 2'-OMe sugar moiety and 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from cEt sugar moiety and LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholinyl, modified morpholinyl, PNA, THP and F-HNA.

[0236] In certain embodiments, the modified oligonucleotide comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleosides comprising modified sugar moieties. In certain embodiments, modified sugar moieties are independently selected from 2'-substituted sugar moieties, bicyclic sugar moieties or sugar surrogates. In certain embodiments, 2'-substituted sugar moieties are selected from 2'-MOE sugar moieties, 2'-NMA sugar moieties, 2'-OMe sugar moieties and 2'-F sugar moieties. In certain embodiments, bicyclic sugar moieties are selected from cEt sugar moieties and LNA sugar moieties. In certain embodiments, sugar surrogates are selected from morpholinyl, modified morpholinyl, THP and F-HNA.

[0237] In certain embodiments, each nucleoside of a modified oligonucleotide comprises a modified sugar moiety ("completely modified oligonucleotide"). In certain embodiments, each nucleoside of a completely modified oligonucleotide comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from 2'-MOE sugar moiety, 2'-NMA sugar moiety, 2'-OMe sugar moiety, and 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from cEt sugar moiety and LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholinyl, modified morpholinyl, THP, and F-HNA. In certain embodiments, each nucleoside of a completely modified oligonucleotide comprises the same modified sugar moiety ("sugar motif modified uniformly"). In certain embodiments, the length of the sugar motif modified uniformly is 7 to 20 nucleosides. In certain embodiments, each nucleoside of a sugar motif modified uniformly comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from 2'-MOE sugar moiety, 2'-NMA sugar moiety, 2'-OMe sugar moiety and 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from cEt sugar moiety and LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholinyl, modified morpholinyl, THP and F-HNA. In certain embodiments, the modified oligonucleotide with at least one fully modified sugar motif may also include at least 1, at least 2, at least 3 or at least 4 2'-deoxyribonucleosides.

[0238] Certain nucleobase motifs

[0239] In certain embodiments, the oligonucleotide comprises modified and / or unmodified nucleobases arranged in a defined pattern or motif along the oligonucleotide or a portion thereof. In certain embodiments, each nucleobase is modified. In certain embodiments, no nucleobase is modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all pyrimidine nucleobases in the modified oligonucleotide are 5-methylcytosine. In certain embodiments, all cytosine nucleobases are 5-methylcytosine, and all other nucleobases of the modified oligonucleotide are unmodified nucleobases.

[0240] In certain embodiments, the modified oligonucleotide comprises a block of modified nucleobases. In certain such embodiments, the block is at the 3' end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides at 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 at the 5' end of the oligonucleotide.

[0241] In certain embodiments, the oligonucleotide with the spacer motif comprises a nucleoside comprising a modified nucleobase. In certain such embodiments, a nucleoside comprising a modified nucleobase is in the central gap of the oligonucleotide with the spacer motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2'-deoxyribosyl sugar moiety. In certain embodiments, the modified nucleobase is selected from: 2-thiopyrimidine and 5-propyne pyrimidine.

[0242] Certain internucleoside linkage motifs

[0243] In certain embodiments, the oligonucleotide comprises modified and / or unmodified internucleoside linkages arranged along the oligonucleotide or its portion in a defined pattern or motif. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is a thiophosphate internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is independently selected from thiophosphate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, each thiophosphate internucleoside linkage is independently selected from stereo-irregular thiophosphate, (Sp) thiophosphate and (Rp) thiophosphate. In certain embodiments, the sugar motif of the modified oligonucleotide is a spacer and the internucleoside linkages in the gap are all modified. In certain such embodiments, some or all internucleoside linkages in the flank are unmodified phosphodiester internucleoside linkages. In certain embodiments, the terminal internucleoside linkages are modified. In certain embodiments, the sugar motif of the modified oligonucleotide is a spacer, and the internucleoside linkage motif includes at least one phosphodiester internucleoside linkage in at least one flank, wherein the at least one phosphodiester internucleoside linkage is not a terminal internucleoside linkage, and the remaining internucleoside linkages are thiophosphate internucleoside linkages. In certain such embodiments, all thiophosphate internucleoside linkages are stereo-irregular. In certain embodiments, all thiophosphate internucleoside linkages in the flank are (Sp) thiophosphates, and the gap includes at least one Sp, Sp, Rp motif. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides comprising such internucleoside linkage motifs. In certain embodiments, one or more internucleoside linkages are methylsulfonyl phosphoramidate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from phosphodiester internucleoside linkages, thiophosphate internucleoside linkages, and methylsulfonyl phosphoramidate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from thiophosphate internucleoside linkage and methylsulfonyl phosphoramidate internucleoside linkage. In certain embodiments, one or more internucleoside linkages are methoxypropylphosphonate internucleoside linkages. In certain embodiments, each internucleoside linkage is independently selected from phosphodiester internucleoside linkage, thiophosphate internucleoside linkage and methoxypropylphosphonate internucleoside linkage. In certain embodiments, each internucleoside linkage is independently selected from thiophosphate internucleoside linkage and methoxypropylphosphonate internucleoside linkage.

[0244] In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphodiester internucleoside linkages. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphorothioate internucleoside linkages. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, or at least 5 phosphodiester internucleoside linkages and the remaining internucleoside linkages are phosphorothioate internucleoside linkages.

[0245] Certain lengths

[0246] It is possible to increase or decrease the length of the oligonucleotide without eliminating activity. For example, in Woolf et al., Proc.Natl.Acad.Sci.USA, 1992,89,7305-7309,1992), a series of oligonucleotides with a length of 13-25 core bases were tested to induce the ability of target nucleic acid cutting in the oocyte injection model. Oligonucleotides with a length of 25 core bases and 8 or 11 mismatched bases near the end of the oligonucleotide can guide the specific cutting of the target nucleic acid, but the degree is lower than the oligonucleotides without mismatching. Similarly, using the oligonucleotides of 13 core bases, including those oligonucleotides with 1 or 3 mismatches, target-specific cutting is achieved.

[0247] In certain embodiments, oligonucleotides (including modified oligonucleotides) may have any one of a variety of length ranges. In certain embodiments, oligonucleotides are composed of X to Y connected nucleosides, wherein X represents the minimum number of nucleosides in the range and Y represents the maximum number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50; Condition is X≤Y. For example, in certain embodiments, the oligonucleotides range from 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27 8, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26,20 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, 2 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.

[0248] In certain embodiments, the oligonucleotide consists of 16 connected nucleosides. In certain embodiments, the oligonucleotide consists of 17 connected nucleosides. In certain embodiments, the oligonucleotide consists of 18 connected nucleosides. In certain embodiments, the oligonucleotide consists of 19 connected nucleosides. In certain embodiments, the oligonucleotide consists of 20 connected nucleosides.

[0249] Certain modified oligonucleotides

[0250] In certain embodiments, the above-mentioned modifications (sugar, core base, internucleoside linkage) are incorporated into modified oligonucleotides. In certain embodiments, the modified oligonucleotides are characterized by their modified motifs and overall length. In certain embodiments, such parameters are independent of each other. Therefore, unless otherwise indicated, each internucleoside linkage of an oligonucleotide with a spacer sugar motif may be modified or unmodified and may or may not follow a sugar-modified spacer modification pattern. For example, the internucleoside linkage in the flanking region of a sugar spacer may be identical or different from each other, and may be identical or different from the internucleoside linkage in the gap region of a sugar motif. Similarly, such sugar spacer oligonucleotides may include one or more modified core bases that are unrelated to the sugar-modified spacer pattern. Unless otherwise indicated, all modifications are unrelated to the core base sequence.

[0251] Populations of certain modified oligonucleotides

[0252] A population of modified oligonucleotides (wherein all modified oligonucleotides of the population have the same molecular formula) can be a stereo-random population or a chiral-enriched population. All chiral centers of all modified oligonucleotides in a stereo-random population are stereo-random. In a chiral-enriched population, at least one specific chiral center in the modified oligonucleotides of the population is not stereo-random. In certain embodiments, the modified oligonucleotides of the chiral-enriched population are enriched in β-D ribosyl sugar moieties, and all thiophosphate nucleoside interlinkages are stereo-random. In certain embodiments, the modified oligonucleotides of the chiral-enriched population are enriched in β-D ribosyl sugar moieties and at least one specific thiophosphate nucleoside interlinkage in a specific stereochemical configuration.

[0253] Nucleobase sequence

[0254] In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by a core base sequence. In certain embodiments, the core base sequence of an oligonucleotide is complementary to a second oligonucleotide or an identified reference nucleic acid (e.g., a target nucleic acid). In certain such embodiments, the core base sequence of a portion of an oligonucleotide is complementary to a second oligonucleotide or an identified reference nucleic acid (e.g., a target nucleic acid). In certain embodiments, the core base sequence of a portion or the 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 a second oligonucleotide or nucleic acid (e.g., a target nucleic acid).

[0255] Certain oligomeric compounds

[0256] In certain embodiments, oligomeric compounds are provided herein, which are composed of oligonucleotides (modified or unmodified) and optionally one or more conjugated groups and / or terminal groups. The conjugated group is composed of one or more conjugated moieties and a conjugated joint connecting the conjugated moiety to the oligonucleotide. The conjugated group can be connected to either end or both ends and / or any internal position of the oligonucleotide. In certain embodiments, the conjugated group is connected to the 2' position of the nucleoside of the modified oligonucleotide. In certain embodiments, the conjugated group connected to either end or both ends of the oligonucleotide is a terminal group. In certain such embodiments, the conjugated group or the terminal group is connected to the 3' end and / or 5' end of the oligonucleotide. In certain such embodiments, the conjugated group (or terminal group) is connected to the 3' end of the oligonucleotide. In certain embodiments, the conjugated group is connected near the 3' end of the oligonucleotide. In certain embodiments, the conjugated group (or terminal group) is connected to the 5' end of the oligonucleotide. In certain embodiments, the conjugated group is connected near the 5' end of the oligonucleotide.

[0257] Examples of terminal groups include, but are not limited to, a conjugating group, a capping group, a phosphate moiety, a protecting group, an abasic nucleoside, a modified or unmodified nucleoside, and two or more independently modified or unmodified nucleosides.

[0258] Certain conjugated groups

[0259] In certain embodiments, the oligonucleotide is covalently linked to one or more conjugated groups. In certain embodiments, the conjugated groups modify one or more properties of the oligonucleotide connected, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cell distribution, cellular uptake, charge and removal. In certain embodiments, the conjugated groups impart new properties to the oligonucleotide connected, for example, a fluorophore or reporter group capable of detecting the oligonucleotide.Certain conjugated groups and conjugated moieties have been described previously, for example: cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYA cad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethyl-ammonium (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783); polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantaneacetic acid palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237); octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937); tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740) or GalNAc cluster (e.g. WO2014 / 179620).

[0260] Conjugated part

[0261] Conjugated moieties include, but are not limited to, intercalators, reporters, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folate, lipids, lipophilic groups, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

[0262] In certain embodiments, the conjugate moiety comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazine, chlorothiazide, diazepam, indomethicin, barbiturate, cephalosporin, sulfonamide, antidiabetic, antibacterial, or antibiotic.

[0263] Conjugation Linker

[0264] The conjugated part is connected to the oligonucleotide via a conjugated joint. In some oligomeric compounds, the conjugated joint is a single chemical bond (i.e., the conjugated part is directly connected to the oligonucleotide via a single bond). In some oligomeric compounds, the conjugated part is connected to the oligonucleotide via a more complex conjugated joint comprising one or more conjugated joint parts, and the one or more conjugated joint parts are the subunits constituting the conjugated joint. In certain embodiments, the conjugated joint comprises an oligomer of a chain structure (e.g., a hydrocarbon chain) or a repeating unit (e.g., ethylene glycol, a nucleoside, or an amino acid unit).

[0265] In certain embodiments, the conjugated joint comprises one or more groups selected from the following: alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino. In certain such embodiments, the conjugated joint comprises a group selected from the following: alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugated joint comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugated joint comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugated joint comprises at least one phosphorus part. In certain embodiments, the conjugated joint comprises at least one phosphate part. In certain embodiments, the conjugated joint includes at least one neutral linking group.

[0266] In certain embodiments, conjugated linkers, including conjugated linkers described above, are bifunctional linking moieties, such as those known in the art that can be used to connect conjugated groups to parent compounds, such as oligonucleotides provided herein. In general, bifunctional linking moieties include at least two functional groups. A functional group is selected to bind to a specific site on the parent compound, and another functional group is selected to bind to the conjugated group. Examples of functional groups used in bifunctional linking moieties include, but are not limited to, electrophilic agents for reacting with nucleophilic groups and nucleophilic agents for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties include one or more groups selected from the following: amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl and alkynyl.

[0267] Examples of conjugated linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic acid (ADO), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugated linkers include, but are not limited to, substituted or unsubstituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Alkynyl, wherein a non-limiting list of preferred substituents includes hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halo, alkyl, aryl, alkenyl, and alkynyl.

[0268] In certain embodiments, the conjugated joint comprises 1-10 joint-nucleosides. In certain embodiments, the conjugated joint comprises 2-5 joint-nucleosides. In certain embodiments, the conjugated joint comprises exactly 3 joint-nucleosides. In certain embodiments, the conjugated joint comprises a TCA motif. In certain embodiments, such joint-nucleosides are modified nucleosides. In certain embodiments, such joint-nucleosides comprise modified sugar moieties. In certain embodiments, joint-nucleosides are unmodified. In certain embodiments, joint-nucleosides comprise optionally protected heterocyclic bases selected from the following: purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable portion is a nucleoside selected from the following: uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is usually desirable to cut the oligomeric compound from the oligomeric compound after the oligomeric compound reaches the target tissue. Therefore, the linker-nucleoside is usually connected to each other and to the remainder of the oligomeric compound by a cleavable bond. In certain embodiments, this type of cleavable bond is a phosphodiester bond.

[0269] In this article, joint-nucleoside is not considered as a part of oligonucleotide. Therefore, in the embodiment that oligomeric compound comprises the nucleoside composition of the connection of the specified number or scope and / or has the oligonucleotide of the specified complementarity percentage with reference nucleic acid and / or oligomeric compound also comprises the conjugated group of the conjugated joint including the conjugated joint with joint-nucleoside, those joint-nucleoside are not counted in the length of oligonucleotide and are not used to determine the complementarity percentage of oligonucleotide for reference nucleic acid. For example, oligomeric compound can comprise (1) the modified oligonucleotide consisting of 8-30 nucleosides and (2) the conjugated group of joint-nucleoside comprising 1-10 continuous with the nucleoside of modified oligonucleotide. The total number of continuous connection nucleosides in such oligomeric compound is more than 30. Alternatively, oligomeric compound can comprise the modified oligonucleotide consisting of 8-30 nucleosides and without conjugated group. The total number of continuous connection nucleosides in such oligomeric compound is no more than 30. Unless otherwise indicated, the conjugated joint comprises no more than 10 joint-nucleosides. In certain embodiments, the conjugated linker comprises no more than 5 linkers-nucleosides. In certain embodiments, the conjugated linker comprises no more than 3 linkers-nucleosides. In certain embodiments, the conjugated linker comprises no more than 2 linkers-nucleosides. In certain embodiments, the conjugated linker comprises no more than 1 linker-nucleoside.

[0270] In certain embodiments, it is desirable to cut the conjugated group from the oligonucleotide. For example, in some cases, the oligomeric compound comprising a specific conjugated portion can be better taken up by a specific cell type, but once the oligomeric compound is taken up, it is desirable to cut the conjugated group to release the unconjugated or parent oligonucleotide. Therefore, some conjugated joints may include one or more cleavable parts. In certain embodiments, the cleavable part is a cleavable bond. In certain embodiments, the cleavable part is a group of atoms comprising at least one cleavable bond. In certain embodiments, the cleavable part includes a group of atoms having one, two, three, four or more than four cleavable bonds. In certain embodiments, the cleavable part is selectively cut in a cell or subcellular compartment such as a lysosome. In certain embodiments, the cleavable part is selectively cut by an endogenous enzyme such as a nuclease.

[0271] In certain embodiments, the cleavable bond is selected from: an amide, an ester, an ether, one or two esters of a phosphodiester, a phosphate, a carbamate, or a disulfide. In certain embodiments, the cleavable bond is one or two esters of a phosphodiester. In certain embodiments, the cleavable portion comprises a phosphate or a phosphodiester. In certain embodiments, the cleavable portion is a phosphate linkage between an oligonucleotide and a conjugated portion or conjugated group.

[0272] In certain embodiments, the cleavable portion comprises or consists of one or more joints-nucleosides. In certain such embodiments, the one or more joints-nucleosides are connected to each other and / or to the remainder of the oligomeric compound by a cleavable bond. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, the cleavable portion is a 2'-deoxyribonucleoside, which is connected to the 3' or 5' terminal nucleoside of the oligonucleotide by a phosphate internucleoside linkage and is covalently connected to the conjugated joint or the remainder of the conjugated portion by a phosphate or thiophosphate internucleoside linkage. In certain such embodiments, the cleavable portion is a 2'-deoxyadenosine.

[0273] Certain terminal groups

[0274] In certain embodiments, the oligomeric compound comprises one or more terminal groups. In certain such embodiments, the oligomeric compound comprises a stabilized 5'-phosphate. Stabilized 5'-phosphate includes but is not limited to 5'-phosphonates, including but not limited to 5'-vinylphosphonates. In certain embodiments, the terminal group comprises one or more abasic nucleosides and / or reverse nucleosides. In certain embodiments, the terminal group comprises one or more 2'-connected nucleosides. In certain such embodiments, the 2'-connected nucleosides are abasic nucleosides.

[0275] Oligoduplex

[0276] In certain embodiments, the oligomeric compound described herein comprises an oligonucleotide whose core base sequence is complementary to the sequence of the target nucleic acid. In certain embodiments, the oligomeric compound is paired with the second oligomeric compound to form an oligomeric duplex. Such oligomeric duplexes include a first oligomeric compound complementary to the target nucleic acid and a second oligomeric compound complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of the oligomeric duplex comprises the following or is composed of the following: (1) a modified or unmodified oligonucleotide and optionally a conjugated group and (2) a second modified or unmodified oligonucleotide and optionally a conjugated group. Any one or two oligomeric compounds of the oligomeric duplex may include a conjugated group. The oligonucleotide of each oligomeric compound of the oligomeric duplex may include non-complementary overhanging nucleosides.

[0277] Antisense activity

[0278] In certain embodiments, oligomeric compounds and oligomeric duplexes are capable of hybridizing with target nucleic acids to produce at least one antisense activity; such oligomeric compounds and oligomeric duplexes are antisense compounds. In certain embodiments, when the antisense compound reduces, regulates or increases the amount or activity of the target nucleic acid by 25% or more in a standard cell assay, it has antisense activity. In certain embodiments, the antisense compound selectively affects one or more target nucleic acids. Such antisense compounds include the following nuclear base sequence, which hybridizes with one or more target nucleic acids, produces one or more desired antisense activities, and does not hybridize with one or more non-target nucleic acids, or does not hybridize with one or more non-target nucleic acids in a manner that produces significant non-desired antisense activity.

[0279] In certain antisense activities, hybridization of the antisense compound to the target nucleic acid results in the recruitment of proteins that cleave 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 this RNA:DNA duplex need not be unmodified DNA. In certain embodiments, provided herein are antisense compounds that are sufficiently "DNA-like" to trigger RNase H activity. In certain embodiments, one or more non-DNA-like nucleosides in the gap between the spacers are tolerated.

[0280] In some antisense activities, the antisense compound or a portion of the antisense compound is loaded into the RNA-induced silencing complex (RISC), ultimately leading to target nucleic acid cleavage. For example, some antisense compounds lead to target nucleic acid cleavage through Argonaute. The antisense compound loaded into RISC is an RNAi compound. The RNAi compound can be double-stranded (siRNA) or single-stranded (ssRNA).

[0281] In certain embodiments, the hybridization of antisense compounds and target nucleic acid does not result in the recruitment of proteins that cut the target nucleic acid. In certain embodiments, the hybridization of antisense compounds and target nucleic acid results in the change of the splicing of target nucleic acid. In certain embodiments, the hybridization of antisense compounds and target nucleic acid results in the inhibition of the binding interaction between target nucleic acid and protein or other nucleic acids. In certain embodiments, the hybridization of antisense compounds and target nucleic acid results in the change of the translation of target nucleic acid. In certain embodiments, the hybridization of antisense compounds and target nucleic acid results in exon inclusion. In certain embodiments, the hybridization of antisense compounds and target nucleic acid results in the amount or activity increase of target nucleic acid. In certain embodiments, the hybridization of antisense compounds complementary to target nucleic acid results in the change of splicing, thereby resulting in the inclusion of exons in mRNA.

[0282] Antisense activity can be observed directly or indirectly. In certain embodiments, observing or detecting antisense activity involves observing or detecting a change in the amount of a target nucleic acid or a protein encoded by the 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.

[0283] Certain target nucleic acids

[0284] In certain embodiments, the oligomeric compound comprises or consists of a portion of oligonucleotides complementary to the target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: mature mRNA and pre-mRNA, including introns, exons and untranslated regions. In certain embodiments, the target nucleic acid is mature mRNA. In certain embodiments, the target nucleic acid is 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, at least 50% of the target region is within an intron.

[0285] Complementary to target nucleic acid / Mismatch

[0286] 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 oligonucleotides with 100% complementarity to bcl-2 mRNA and 3 mismatches to bcl-xL mRNA to reduce the expression of bcl-2 and bcl-xL in vivo and in vitro. In addition, this oligonucleotide showed effective anti-tumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16: 3341-3358, 1988) tested a series of 14-nucleobase oligonucleotides in series and 28 and 42-nucleobase oligonucleotides containing sequences of two or three tandem oligonucleotides, respectively, in rabbit reticulocyte assays to inhibit the translation of human DHFR. Three 14-nucleobase oligonucleotides were each individually able to inhibit translation, but at a more moderate level compared to 28 or 42-nucleobase oligonucleotides.

[0287] In certain embodiments, the oligonucleotide is complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, the oligonucleotide is 99%, 95%, 90%, 85% or 80% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and a portion is 100% or fully complementary to the target nucleic acid. In certain embodiments, the length of the fully complementary portion is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core bases.

[0288] In certain embodiments, the oligonucleotide comprises one or more mismatched nucleobases relative to the target nucleic acid. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 from the 5' end of the oligonucleotide.

[0289] SMN2

[0290] In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide complementary to a target nucleic acid encoding SMN2 or a portion thereof. In certain embodiments, SMN2 has the sequence shown in SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14, truncated from nucleotides 19939708 to 19967777).

[0291] In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO: 1 modulates splicing of SMN2 RNA in the cell. In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO: 1 increases the amount of SMN2 RNA including exon 7. In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO: 1 increases expression of full-length SMN2 protein. In certain embodiments, the oligomeric compound consists of a modified oligonucleotide.

[0292] In certain embodiments, contacting a subject's cells with an oligomeric compound complementary to SEQ ID NO: 1 improves one or more symptoms of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is SMA, including type I SMA, type II SMA, type III SMA, and type IV SMA. In certain embodiments, the symptom is any one of: decreased muscle strength; inability to sit upright, stand, and / or walk or reduced ability; decreased neuromuscular activity; decreased electrical activity in one or more muscles; decreased respiration; inability to eat, drink, and / or breathe without assistance or reduced ability; weight loss or decreased weight gain; and / or decreased survival.

[0293] In certain embodiments, an oligomeric compound complementary to SEQ ID NO: 1 is capable of increasing SMN2 RNA including exon 7 by at least 1-fold, 2-fold, or 3-fold in vivo 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 by at least 1-fold, 2-fold, or 3-fold in vivo when administered according to a standard in vivo assay.

[0294] Certain target nucleic acids in certain tissues

[0295] In certain embodiments, the oligomeric compound comprises or consists of a portion of oligonucleotides complementary to the target nucleic acid, wherein the target nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the pharmacologically relevant tissue is a cell and tissue constituting the central nervous system (CNS). Such tissues include brain tissue, such as spinal cord, cortex and coronary brain tissue.

[0296] Certain pharmaceutical compositions

[0297] In certain embodiments, pharmaceutical compositions comprising one or more oligomeric compounds are described herein. In certain embodiments, the one or more oligomeric compounds are each composed of modified oligonucleotides. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises a sterile saline solution and one or more oligomeric compounds or consists of it. In certain embodiments, sterile saline is pharmaceutical grade saline. In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and sterile water or consists of it. In certain embodiments, sterile water is pharmaceutical grade water. In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and phosphate buffered saline (PBS) or consists of it. In certain embodiments, sterile PBS is pharmaceutical grade PBS. In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and artificial cerebrospinal fluid ("artificial CSF" or "aCSF") or consists of it. In certain embodiments, artificial cerebrospinal fluid is pharmaceutical grade.

[0298] In certain embodiments, the pharmaceutical composition comprises a modified oligonucleotide and an artificial cerebrospinal fluid. In certain embodiments, the pharmaceutical composition consists of a modified oligonucleotide and an artificial cerebrospinal fluid. In certain embodiments, the pharmaceutical composition consists essentially of a modified oligonucleotide and an artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

[0299] In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and one or more excipients. In certain embodiments, the excipient is selected from water, saline, ethanol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinyl pyrrolidone.

[0300] In certain embodiments, the oligomeric compound can be mixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions or formulations. The composition and method of formulation of the pharmaceutical composition depends on many criteria, including but not limited to the route of administration, the extent of the disease or the dose to be administered.

[0301] In certain embodiments, the pharmaceutical composition comprising an oligomeric compound encompasses any pharmaceutically acceptable salt of the oligomeric compound, an ester of the oligomeric compound, or a salt of such an ester. In certain embodiments, the pharmaceutical composition comprising an oligomeric compound including one or more oligonucleotides, when applied to a subject (including humans), can provide (directly or indirectly) a biologically active metabolite or its residue. Therefore, for example, the disclosure also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs and other bioequivalents of oligomeric compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In certain embodiments, the prodrug comprises one or more conjugated groups connected to an oligonucleotide, wherein the conjugated group is cut in vivo by an endogenous nuclease. In certain embodiments, the prodrug comprises one or more conjugated groups connected to an oligonucleotide, wherein the conjugated group is cut in vivo by an endogenous nuclease.

[0302] Lipid moieties have been used in nucleic acid therapy in a variety of methods. In some such methods, nucleic acids (e.g., oligomeric compounds) are introduced into preformed liposomes or lipid complexes (lipoplexes) made of a mixture of cationic lipids and neutral lipids. In some methods, DNA complexes with monocationic lipids or polycationic lipids are formed in the absence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceutical agents to specific cells or tissues. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceutical agents to adipose tissue. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceutical agents to muscle tissue.

[0303] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including pharmaceutical compositions comprising hydrophobic compounds. In certain embodiments, certain organic solvents are used, such as dimethyl sulfoxide.

[0304] In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents comprising oligomeric compounds provided herein to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with tissue-specific antibodies.

[0305] In certain embodiments, the pharmaceutical composition comprises a co-solvent system. Some of 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 a VPD co-solvent system, which comprises 3% w / v benzyl alcohol, 8% w / v non-polar surfactant polysorbate 80, and 10% w / v ethanol. TM and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such cosolvent systems can be varied significantly without significantly changing their solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components can be varied: for example, other surfactants can be used in place of polysorbate 80. TM ; The fraction size of polyethylene glycol can vary; other biocompatible polymers can replace polyethylene glycol, for example, polyvinyl pyrrolidone; and other sugars or polysaccharides can replace dextrose.

[0306] In certain embodiments, the pharmaceutical composition is prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.). In certain such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution, the aqueous solution being, for example, water or a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or a saline buffer. In certain embodiments, other ingredients (e.g., ingredients that help dissolve or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. Some pharmaceutical compositions for injection are present in, for example, ampoules or in multidose containers in unit dosage form. Some pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain preparatants, such as suspending agents, stabilizers, and / or dispersants. Certain solvents suitable for use in injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil; synthetic fatty acid esters, such as ethyl oleate or triglycerides; and liposomes.

[0307] Under certain conditions, some compounds disclosed herein act as acids. Although such compounds can be drawn or described in the form of protonation (free acid) or ionization and association with cations (salt), the aqueous solution of such compounds exists in such forms in equilibrium. For example, the phosphate linkage of oligonucleotides in aqueous solution exists in equilibrium in the form of free acid, anion and salt. Unless otherwise indicated, the compounds described herein are intended to include all such forms. In addition, some oligonucleotides have several such linkages, each of which is balanced. Therefore, the oligonucleotides in the solution exist in multiple positions in a series of forms, all in equilibrium. The term "oligonucleotide" is intended to include all such forms. The drawn structure must depict a single form. However, unless otherwise indicated, such drawings are also intended to include corresponding forms. In this article, the structure of the free acid of the depicted compound is followed by the term "or its salt" to explicitly include all such forms that may be fully or partially protonated / deprotonated / associated with cations. In some cases, one or more specific cations are identified.

[0308] In certain embodiments, the modified oligonucleotide or oligomeric compound is in an aqueous solution containing sodium. In certain embodiments, the modified oligonucleotide or oligomeric compound is in an aqueous solution containing potassium. In certain embodiments, the modified oligonucleotide or oligomeric compound is in PBS. In certain embodiments, the modified oligonucleotide or oligomeric compound is in water. In certain such embodiments, the pH value of the solution is adjusted with NaOH and / or HCl to reach the desired pH value.

[0309] In this article, some specific dosages are described. Dosage can be in dosage unit form. For clarity, the dosage (or dosage unit) (mg) of modified oligonucleotide or oligomeric compound indicates the mass of modified oligonucleotide or oligomeric compound in free acid form. As described above, in aqueous solution, free acid is balanced with anion and salt form. However, for the purpose of calculating dosage, it is assumed that modified oligonucleotide or oligomeric compound exists in solvent-free, sodium acetate-free, anhydrous, free acid form. For example, in the case of modified oligonucleotide or oligomeric compound in a solution (such as saline) comprising sodium, modified oligonucleotide or oligomeric compound can be partially or completely deprotonated and associated with Na+ ions. However, the mass of protons is still included in the weight of dosage, and the mass of Na+ ions is not included in the weight of dosage. 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 is equivalent to: 10.53 mg of solvent-free, sodium acetate-free, anhydrous sodium ionized Compound No. 1263789; 10.53 mg of solvent-free, sodium acetate-free, anhydrous sodium ionized Compound No. 1287717; 10.52 mg of solvent-free, sodium acetate-free, anhydrous sodium ionized Compound No. 1287745; and 10.51 mg of solvent-free, sodium acetate-free, anhydrous sodium ionized Compound No. 1358996. When an oligomeric compound contains a conjugated group, the mass of the conjugated group is included in calculating the dose of such oligomeric compound. If the conjugated group also has an acid, the conjugated group is also assumed to be fully protonated for the purpose of calculating the dose.

[0310] Certain compositions

[0311] Compound number: 1263789

[0312] In certain embodiments, compound No. 1263789 is characterized as a modified oligonucleotide having the sequence (5' to 3')CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a 2'-MOE sugar moiety, wherein the internucleoside linkages between nucleosides 2 to 3 and 4 to 5 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0313] In certain embodiments, Compound No. 1263789 is represented by the following chemical notation (5' to 3'): m C es A eo m C es T eo T es T es m C es A es T es A es A es T es G es m C es T es G es G es m C e (SEQ ID NO:21),

[0314] in,

[0315] A = adenine nucleobase,

[0316] m C=5-methylcytosine nucleobase,

[0317] G = guanine nucleobase,

[0318] T = thymine nucleobase,

[0319] e=2'-MOE sugar moiety,

[0320] s = phosphorothioate internucleoside linkage, and

[0321] o = phosphodiester internucleoside linkage.

[0322] In certain embodiments, Compound No. 1263789 is represented by the following chemical structure:

[0323]

[0324] (SEQ ID NO:21).

[0325] Structure 1. Compound No. 1263789

[0326] In certain embodiments, the sodium salt of Compound No. 1263789 is represented by the following chemical structure:

[0327]

[0328] (SEQ ID NO:21).

[0329] Structure 2. Sodium salt of compound number 1263789

[0330] Compound number: 1287717

[0331] In certain embodiments, compound No. 1287717 is characterized as a modified oligonucleotide having the sequence (5' to 3')TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each nucleoside comprises a 2'-MOE sugar moiety, wherein the internucleoside linkages between nucleosides 1 to 2 and 19 to 20 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, and 18 to 19 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0332] In certain embodiments, Compound No. 1287717 is represented by the following chemical notation (5' to 3'):

[0333] T eo T es m C es A es m C es T es T es T es m C es A es T es A es A es T es G es m C es T es G es G eo m C e (SEQ ID NO:22)

[0334] in,

[0335] A = adenine nucleobase,

[0336] m C=5-methylcytosine nucleobase,

[0337] G = guanine nucleobase,

[0338] T = thymine nucleobase,

[0339] e=2'-MOE sugar moiety,

[0340] s = phosphorothioate internucleoside linkage, and

[0341] o = phosphodiester internucleoside linkage.

[0342] In certain embodiments, Compound No. 1287717 is represented by the following chemical structure:

[0343]

[0344] (SEQ ID NO:22).

[0345] Structure 3. Compound No. 1287717

[0346] In certain embodiments, the sodium salt of Compound No. 1287717 is represented by the following chemical structure:

[0347]

[0348] (SEQ ID NO:22).

[0349] Structure 4. Sodium salt of compound number 1287717

[0350] Compound number: 1287745

[0351] In certain embodiments, compound No. 1287745 is characterized as a modified oligonucleotide having the sequence (5' to 3')TTCACTTTCATAATGCTGGC (SEQ ID NO: 22), wherein each of nucleosides 1 and 20 comprises a 2'-MOE sugar moiety, each of nucleosides 2-19 comprises a 2'-NMA sugar moiety, wherein the internucleoside linkages between nucleosides 1 to 2 and 19 to 20 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, and 18 to 19 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0352] In certain embodiments, Compound No. 1287745 is represented by the following chemical notation (5' to 3'):

[0353] T eo T ns m Cns A ns m C ns T ns T ns T ns m C ns A ns T ns A ns A ns T ns G ns m C ns T ns G ns G no m C e (SEQ ID NO:22)

[0354] in,

[0355] A = adenine nucleobase,

[0356] m C=5-methylcytosine nucleobase,

[0357] G = guanine nucleobase,

[0358] T = thymine nucleobase,

[0359] e=2'-MOE sugar moiety,

[0360] n=2'-NMA sugar moiety,

[0361] s = phosphorothioate internucleoside linkage, and

[0362] o = phosphodiester internucleoside linkage.

[0363] In certain embodiments, Compound No. 1287745 is represented by the following chemical structure:

[0364]

[0365] (SEQ ID NO:22).

[0366] Structure 5. Compound No. 1287745

[0367] In certain embodiments, the sodium salt of Compound No. 1287745 is represented by the following chemical structure:

[0368]

[0369] (SEQ ID NO:22).

[0370] Structure 6. Sodium salt of compound number 1287745

[0371] Compound No.: 1358996

[0372] In certain embodiments, compound No. 1358996 is characterized as a modified oligonucleotide having the sequence (5' to 3')CACTTTCATAATGCTGGC (SEQ ID NO: 21), wherein each nucleoside comprises a 2'-NMA sugar moiety, wherein the internucleoside linkages between nucleosides 2 to 3 and 4 to 5 are phosphodiester internucleoside linkages, and the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is 5-methylcytosine.

[0373] In certain embodiments, Compound No. 1358996 is represented by the following chemical notation (5' to 3'):

[0374] m C ns A no m C ns T no T ns T ns m C ns A ns T ns A ns A ns T ns G ns m C ns T ns G ns G ns m C n (SEQ ID NO:21)

[0375] in,

[0376] A = adenine nucleobase,

[0377] m C=5-methylcytosine nucleobase,

[0378] G = guanine nucleobase,

[0379] T = thymine nucleobase,

[0380] n=2'-NMA sugar moiety,

[0381] s = phosphorothioate internucleoside linkage, and

[0382] o = phosphodiester internucleoside linkage.

[0383] In certain embodiments, Compound No. 1358996 is represented by the following chemical structure:

[0384]

[0385] (SEQ ID NO:21).

[0386] Structure 7. Compound No. 1358996

[0387] In certain embodiments, the sodium salt of Compound No. 1358996 is represented by the following chemical structure:

[0388]

[0389] (SEQ ID NO:21).

[0390] Structure 8. Sodium salt of compound number 1358996

[0391] Certain comparative compositions

[0392] In certain embodiments, an agent approved for the treatment of SMA (generic name nusinersen; compound number 396443) is a comparative compound (see, for example, Chiroboga et al., Neurology, 86(10):890-897, 2016; Finkel et al., Lancet, 338(10063):3017-3026, 2016; Finkel et al., N. Engl. J. Med., 377(18):1723-17322017; Mercuri et al., N. Engl. J. Med., 378(7):625-635, 2018; Montes et al., Muscle Nerve. 60(4):409-414, 2019; Darras et al., Neurology, 92(21):e2492-e2506, 2019). Previously described in WO2010120820 (which is incorporated herein by reference) and having the sequence (5' to 3')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 5-methylcytosine.

[0393] In certain embodiments, other previously described compounds, including Compound Nos. 387954, 396442, 443305, and 819735, are comparative compounds, although not approved for use in human therapy.

[0394] Compound No. 387954 was previously described in WO2014 / 179620, which is incorporated herein by reference. Compound No. 387954 has the sequence (5' to 3')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.

[0395] Compound No. 396442 was previously described in WO 2010 / 120820, which is incorporated herein by reference. Compound No. 396442 has the sequence (5' to 3')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 5-methylcytosine.

[0396] 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')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.

[0397] 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') 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.

[0398] Table 1

[0399] Certain comparative compositions

[0400]

[0401] In certain embodiments, the compounds described herein are superior to the compounds described in WO 2007 / 002390, WO 2010 / 120820, WO 2015 / 161170, and WO 2018 / 014041 in that they exhibit one or more improved properties, such as potency, efficacy, and tolerability.

[0402] For example, Compound No. 1263789, Compound No. 1287745, and Compound No. 1358996 each exhibited improved in vivo efficacy compared to Compound No. 396443. As shown in Example 5, Compound No. 1263789, Compound No. 1287745, and Compound No. 1358996 achieved ED values ​​of 13.3, 8.8, and 7.4 in the spinal cord, respectively. 50 In contrast, compound number 396443 achieved an ED of 22.0 in the spinal cord. 50 Thus, each of Compound No. 1263789, Compound No. 1287745, and Compound No. 1358996 were more potent than Compound No. 396443 in this assay.

[0403] For example, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 each exhibited an improved 3-hour FOB score compared to Compound No. 396443, Compound No. 387954, and Compound No. 443305. As shown in Example 6, at 700 μg, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 achieved 3-hour FOB scores of 0, 3.25, 1, and 0, respectively. In contrast, at half the dose (350 μg), Compound No. 396443 achieved a 3-hour FOB score of 4.0; and at the same dose (700 μg), Compound No. 387954 and Compound No. 443305 achieved 3-hour FOB scores of 4.0 and 4.75, respectively. Thus, each of Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 was better tolerated than Compound No. 396443, Compound No. 387954, and Compound No. 443305 in this assay.

[0404] For example, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 each exhibit improved long-term tolerability compared to Compound No. 396442 and Compound No. 819735. As shown in Example 7, Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 showed no adverse effects, no Purkinje cell loss, and cortical GFAP mRNA less than 2 times the control. In contrast, 396442 and 819735 each exhibited adverse events, Purkinje cell loss, and cortical GFAP mRNA greater than 2 times the control in certain treated animals. Thus, each of Compound No. 1263789, Compound No. 1287717, Compound No. 1287745, and Compound No. 1358996 were better tolerated than Compound No. 396442 and Compound No. 819735 in this assay.

[0405] NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

[0406] The documents and patents listed herein are openly incorporated herein by reference in their entirety. Although certain compounds, compositions and methods described herein have been particularly described according to certain embodiments, the following examples are only used to illustrate compounds described herein and are not intended to be limited thereto. Each of the references, GenBank accession numbers, etc. cited in this application are incorporated herein by reference in their entirety.

[0407] Although the sequence table accompanying this application identifies each sequence as "RNA" or "DNA" as required, in fact, those sequences can be modified with any combination of chemical modifications. It will be readily understood by those skilled in the art that such nomenclatures describing modified oligonucleotides, such as "RNA" or "DNA", are arbitrary in some cases. For example, an oligonucleotide comprising a nucleoside including a 2'-OH sugar moiety and a thymine base may be described as a DNA with a modified sugar moiety (2'-OH replaces one 2'-H of DNA) or as an RNA with a modified base (thymine (methylated uracil) replaces the uracil of RNA). Therefore, the nucleic acid sequences provided herein, including but not limited to the nucleic acid sequences in the sequence table, are intended to cover nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids with modified nucleobases. By way of further example and without limitation, an oligomeric compound having a nucleobase sequence of "ATCGATCG" encompasses any oligomeric compound having such a nucleobase sequence, whether modified or unmodified, including but not limited to: such compounds comprising RNA bases, such as a sequence 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 "ATCGATCG". m CGAUCG", where m C represents a cytosine base containing a methyl group at the 5-position.

[0408] Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric centers and thus produce enantiomers, diastereomers, and other stereoisomeric configurations, which can be defined according to absolute stereochemistry as (R) or (S), α or β (e.g., for sugar mutarotomers), or (D) or (L) (e.g., for amino acids), etc. Compounds drawn or described as having certain stereoisomeric configurations provided herein include only the indicated compounds. Unless otherwise indicated, compounds drawn or described as having indeterminate stereochemistry provided herein include all such possible isomers, including their stereo-random and optically pure forms. Similarly, unless otherwise indicated, all cis and trans isomers and tautomeric forms of the compounds herein are also included. Oligomeric compounds described herein include chiral pure or chiral enriched mixtures and racemic mixtures. For example, oligomeric compounds having multiple thiophosphate nucleoside linkages include such compounds in which the chirality of the thiophosphate nucleoside linkages is controlled or random. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.

[0409] The compounds described herein include variations in which one or more atoms are replaced by non-radioactive isotopes or radioactive isotopes of the indicated elements. For example, a compound herein containing a hydrogen atom encompasses each 1 All possible deuterium substitutions of H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but are not limited to: 2 H or 3 H instead 1 H; 13 C or 14 C instead 12 C; 15 N instead 14 N; 17 O or 18 O instead 16 O; and 33 S. 34 S. 35 S or 36 S instead 32 S. In certain embodiments, non-radioactive isotope substitution can impart novel properties to oligomeric compounds that are useful as therapeutic or research tools. In certain embodiments, radioactive isotope substitution can render the compounds suitable for research or diagnostic purposes, such as imaging.

[0410] Example

[0411] The following examples illustrate certain embodiments of the present disclosure but are not limiting.In addition, where specific embodiments are provided, the inventors contemplate the universal application of those specific embodiments.

[0412] Example 1: Design of modified oligonucleotides complementary to human SMN2 nucleic acid

[0413] Modified oligonucleotides complementary to human SMN2 nucleic acid were designed and synthesized as indicated in the table below.

[0414] The length of the modified oligonucleotides in the following table is 16, 17, 18, 19 or 20 nucleosides, as specified. The modified oligonucleotides contain 2'-MOE sugar moieties, 2'-NMA sugar moieties, cEt sugar moieties, 2'-OMe sugar moieties and / or 2'-β-D-deoxyribosyl sugar moieties, as specified. Each internucleoside linkage of the entire modified oligonucleotide is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage, as specified. Cytosine is a non-methylated cytosine or a 5-methylcytosine, as specified.

[0415] Unless otherwise specifically stated, each modified oligonucleotide listed in the table below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14, truncated from nucleotides 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column as Underline, bold, italic fonts Each modified oligonucleotide listed in the table below targets the active site of the SMN2 transcript to include exon 7. "Start site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. "Stop site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0416] Table 2

[0417] The length of the modified oligonucleotide in the following table 2 is 16, 17, 18, 19 or 20 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, wherein each 'e' represents a 2'-MOE sugar moiety. Each internucleoside linkage is a thiophosphate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, wherein each 's' represents a thiophosphate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is a 5-methylcytosine.

[0418] Unless otherwise specifically stated, 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). Non-complementary nucleobases are indicated in the nucleobase sequence column as Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0419] Table 2

[0420] 2'-MOE modified oligonucleotides with PS or mixed PS / PO internucleoside linkages

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430] Table 3

[0431] The length of the modified oligonucleotides in Table 3 below is 16, 17, 18, 19 or 20 nucleosides. Each nucleoside comprises a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, wherein each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, wherein each 's' represents a phosphorothioate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0432] 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). "Starting site" indicates the most 5'-nucleoside complementary to the modified oligonucleotide in the target nucleic acid sequence. "Stop site" indicates the most 3'-nucleoside complementary to the modified oligonucleotide in the target nucleic acid sequence.

[0433] Table 3

[0434] 2'-NMA modified oligonucleotides with PS or mixed PS / PO internucleoside linkages

[0435]

[0436]

[0437]

[0438] Table 4

[0439] The length of the modified oligonucleotides in Table 4 below is 18 or 19 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, wherein each 'e' represents a 2'-MOE sugar moiety, and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a thiophosphate internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, wherein each 's' represents a thiophosphate internucleoside linkage. Each cytosine is 5-methylcytosine.

[0440] Unless otherwise specifically stated, 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). Non-complementary nucleobases are indicated in the nucleobase sequence column as Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0441] Table 4

[0442] Mixed 2'-MOE / 2'-NMA modified oligonucleotides with PS internucleoside linkages

[0443]

[0444] Table 5

[0445] The length of the modified oligonucleotides in Table 5 below is 16, 17 or 18 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety or a cEt sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, wherein each 'e' represents a 2'-MOE sugar moiety, and each 'k' represents a cEt sugar moiety. Each internucleoside linkage is a thiophosphate internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, wherein each 's' represents a thiophosphate internucleoside linkage. Each cytosine is a 5-methylcytosine.

[0446] 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). "Starting site" indicates the 5'-nucleoside most complementary to the modified oligonucleotide in the target nucleic acid sequence. "Stop site" indicates the 3'-nucleoside most complementary to the modified oligonucleotide in the target nucleic acid sequence.

[0447] Table 5

[0448] Mixed 2'-MOE / cEt modified oligonucleotides with PS internucleoside linkages

[0449]

[0450]

[0451]

[0452] Table 6

[0453] The length of the modified oligonucleotides in Table 6 below is 19 or 20 nucleosides. 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 of each modified oligonucleotide is provided in the Sugar Motif column, wherein 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 linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): sssssssssssssssssso; wherein each 's' represents a phosphorothioate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Cytosine is either unmethylated cytosine or 5-methylcytosine, wherein each lowercase letter 'c' in the Nucleobase Sequence column represents an unmethylated cytosine, and each uppercase letter 'C' in the Nucleobase Sequence column represents a 5-methylcytosine.

[0454] Unless otherwise specifically stated, 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). Non-complementary nucleobases are indicated in the nucleobase sequence column with Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0455] Table 6

[0456] Modified oligonucleotides with mixed PS / PO internucleoside linkages

[0457]

[0458]

[0459] Table 7

[0460] 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 of each modified oligonucleotide is provided in the Sugar Motif column, wherein each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): ssssssssssssssssoo; wherein each 's' represents a phosphorothioate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0461] Unless otherwise specifically stated, 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). Non-complementary nucleobases are indicated in the nucleobase sequence column with Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0462] Table 7

[0463] Modified oligonucleotides with mixed PS / PO internucleoside linkages

[0464]

[0465]

[0466]

[0467] Table 8

[0468] The length of the modified oligonucleotides in Table 8 below is each 19 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar motif of each modified oligonucleotide is provided in the Sugar Motif column, wherein each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): ssssssssssssososso; wherein each 's' represents a phosphorothioate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0469] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 8 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14, truncated from nucleotides 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column with Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0470] Table 8

[0471] Modified oligonucleotides with mixed PS / PO internucleoside linkages

[0472]

[0473] Table 9

[0474] The length of the modified oligonucleotides in Table 9 below is each 19 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, or a 2'-β-D-deoxyribosyl sugar moiety. The sugar motif of each modified oligonucleotide is provided in the Sugar Motif column, wherein each 'e' represents a 2'-MOE sugar moiety, each 'n' represents a 2'-NMA sugar moiety, and each 'd' represents a 2'-β-D-deoxyribosyl sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): ssssssssssssssosso; wherein each 's' represents a phosphorothioate internucleoside linkage, and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0475] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 9 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14, truncated from nucleotides 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column with Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0476] Table 9

[0477] Modified oligonucleotides with mixed PS / PO internucleoside linkages

[0478]

[0479] Table 10

[0480] The length of the modified oligonucleotides in Table 10 below is each 19 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the Sugar Motif column, wherein each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): ossssssssssssssss; wherein each 's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0481] Unless otherwise specifically stated, each nucleobase in the modified oligonucleotides listed in Table 10 below is complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14, truncated from nucleotides 19939708 to 19967777). Non-complementary nucleobases are indicated in the nucleobase sequence column with Underline, bold, italic fonts The "starting site" indicates the 5'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide. The "ending site" indicates the 3'-most nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0482] Table 10

[0483] Modified oligonucleotides with mixed PS / PO internucleoside linkages

[0484]

[0485] Table 11

[0486] The length of the modified oligonucleotides in Table 11 below is 20 nucleosides each. Each nucleoside comprises a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the Sugar Motif column, wherein each 'e' represents a 2'-MOE sugar moiety and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide provided in the Internucleoside Linkage Motif column is (5' to 3'): ossssssssssssssssso; wherein each 's' represents a phosphorothioate internucleoside linkage and each 'o' represents a phosphodiester internucleoside linkage. Each cytosine is 5-methylcytosine.

[0487] 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). "Starting site" indicates the most 5'-nucleoside complementary to the modified oligonucleotide in the target nucleic acid sequence. "Stop site" indicates the most 3'-nucleoside complementary to the modified oligonucleotide in the target nucleic acid sequence.

[0488] Table 11

[0489] Modified oligonucleotides with mixed PS / PO internucleoside linkages

[0490]

[0491] Example 2: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, single dose (35 μg)

[0492] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. The Taiwanese strain of SMA III mice was obtained from Jackson Laboratory (Bar Harbor, Maine, USA). These mice lack mouse SMN and are 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 are heterozygous for human SMN2 (mSMN+ / -; hSMN2+ / -; FVB.Cg-Tg (SMN2) 2HungSMN1tm1Hung / J) obtained by breeding HOM / HOM (stock number 00005058) with FVB / NJ (stock number 001800).

[0493] deal with

[0494] Homozygous or heterozygous transgenic mice were grouped in groups of 4 mice. Each mouse received a single ICV bolus of 35 μg of modified oligonucleotide. Comparative compound numbers 387954, 396442, and 396443 were also tested in this assay. One group of 4 mice received PBS as a negative control.

[0495] RNA analysis

[0496] 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: CACCTTCCTTCTTTTTGATTTTGTC, designated herein as SEQ ID NO: 12; probe sequence: TACATGAGTGGCTATCATACT (SEQ ID NO: 13)) was used to determine the exon 7 (exon 7 +The amount of SMN2 RNA was determined by using the primer probe set hSMN2_Sumner68_PPS50481 (forward sequence: CATGGTACATGAGTGGCTATCATACTG (SEQ ID NO: 14); reverse sequence: TGGTGTCATTTAGTGCTGCTCTATG (SEQ ID NO: 15); probe sequence CCAGCATTTCCATATAATAGC (SEQ ID NO: 16)) excluding exon 7 (exon 7 - The total SMN2 RNA level was measured using the primer probe set hSMN2_LTS00935 (forward sequence: CAGGAGGATTCCGTGCTGTT (SEQ ID NO: 17); reverse sequence: CAGTGCTGTATCATCCCAAATGTC, (SEQ ID NO: 18); probe sequence: ACAGGCCAGAGCGAT (SEQ ID NO: 19)).

[0497] Results are presented as fold change in RNA levels normalized to total SMN2 levels relative to PBS control. Each of Tables 12-18 represents a different experiment.

[0498] Table 12

[0499] Effects of modified oligonucleotides on human SMN2 RNA splicing in homozygous transgenic mice

[0500]

[0501]

[0502] Table 13

[0503] Effects of modified oligonucleotides on human SMN2 RNA splicing in homozygous transgenic mice

[0504]

[0505]

[0506] Table 14

[0507] Effects of modified oligonucleotides on human SMN2 RNA splicing in homozygous transgenic mice

[0508]

[0509] Table 15

[0510] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0511]

[0512] Table 16

[0513] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0514]

[0515]

[0516] Table 17

[0517] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0518]

[0519] Table 18

[0520] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0521]

[0522] Example 3: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, single dose (15 μg)

[0523] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2. Comparative compound numbers 396443 and 819735 were also tested in this assay. The transgenic mice were divided into groups of 4 mice per group. 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, the mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as the fold change of RNA levels normalized for total SMN2 levels relative to the PBS control. Each of Tables 19-23 represents a different experiment.

[0524] Table 19

[0525] Effects of modified oligonucleotides on human SMN2 RNA splicing in homozygous transgenic mice

[0526]

[0527] Table 20

[0528] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0529]

[0530] Table 21

[0531] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0532]

[0533] Table 22

[0534] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0535]

[0536] Table 23

[0537] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0538]

[0539] Example 4: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, single dose (70 μg)

[0540] The activity of the modified oligonucleotides in human SMN2 transgenic mice was tested essentially as described above in Example 2. The transgenic mice were divided into groups of 4 mice per group. Each mouse received a single ICV bolus of 70 μg of the modified oligonucleotide. One group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as fold change in RNA levels normalized to total SMN2 levels relative to the PBS control.

[0541] Table 24

[0542] Effects of modified oligonucleotides on human SMN2 RNA splicing in homozygous transgenic mice

[0543]

[0544] Example 5: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, multiple doses

[0545] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2. Comparative compound No. 396443 was also tested in this assay. The transgenic mice were divided into groups of 4 mice per group. Each mouse received a single ICV bolus of the modified oligonucleotide at multiple doses as indicated in the following table. One group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the coronary brain and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as the fold change of RNA levels normalized to total SMN2 levels relative to the PBS control. In GraphPad Prism 7, nonlinear regression 4-parameter dose-response curves [Y = bottom + (top - bottom) / (1 + (10^logEC50 / X)^Hill slope)] were used to calculate exon inclusion (exon 7 + ) 50 .

[0546] Table 25

[0547] Effects of modified oligonucleotides on human SMN2 RNA splicing in homozygous transgenic mice

[0548]

[0549] Example 6: Tolerance of modified oligonucleotides complementary to SMN2 in wild-type mice, 3-hour study

[0550] In wild-type female C57 / Bl6 mice, the modified oligonucleotide described above is tested to assess tolerance. Wild-type female C57 / Bl6 mice each receive a single ICV dose of the modified oligonucleotide listed in the table below of 700 μg. Comparative compound No. 396443 was also tested with a dosage of 350 μg in this assay. Comparative compound Nos. 387954, 396442, 443305 and 819735 were also tested with a dosage of 700 μg in this assay. Each treatment group is composed of 4 mice. One group of 4 mice receives PBS as the negative control (identified in the following separate table) for each experiment. 3 hours after injection, mice were evaluated according to seven different standards. The criteria are (1) the mouse is intelligent, alert and responsive; (2) the mouse stands or arches its back without stimulation; (3) the mouse shows no movement without stimulation; (4) the mouse exhibits forward movement after it is lifted; (5) the mouse exhibits no movement after it is lifted; (6) the mouse responds to tail pinch; (7) the mouse breathes evenly. For each of the 7 criteria, if the mouse meets the criteria, the mouse is given a sub-score of 0, and if it does not meet the criteria, a sub-score of 1 (Functional Observation Experiment Composite Score or FOB) is given. After evaluating all 7 criteria, the scores are summed and averaged within each treatment group. The results are presented in the table below. Each of Tables 26-49 represents a different experiment.

[0551] Table 26

[0552] Tolerability score in mice at 350 μg dose

[0553]

[0554] Table 27

[0555] Tolerability score in mice at 700 μg dose

[0556]

[0557] Table 28

[0558] Tolerability score in mice at 700 μg dose

[0559]

[0560]

[0561] Table 29

[0562] Tolerability score in mice at 700 μg dose

[0563]

[0564]

[0565] Table 30

[0566] Tolerability score in mice at 700 μg dose

[0567] Compound No. 3 hours FOB PBS 0.00 396442 3.25 1212961 0.00 1212963 1.00 1212964 2.00 1212965 1.25 1212966 1.25 1212968 0.00 1212971 1.00 1212972 3.25 1212973 0.50 1212974 2.00 1212975 0.50 1212976 1.75

[0568] Table 31

[0569] Tolerability score in mice at 700 μg dose

[0570]

[0571]

[0572] Table 32

[0573] Tolerability score in mice at 700 μg dose

[0574] Compound No. 3 hours FOB PBS 0.00 1212993 7.00 1212994 6.50 1212995 4.25 1212996 3.25 1212997 4.00 1212998 2.00 1212999 1.00 1213000 1.25 1213001 3.00 1213002 2.00 1213003 4.00 1213004 3.00 1213005 3.75 1213006 4.00 1213007 4.00 1213008 3.50

[0575] Table 33

[0576] Tolerability score in mice at 700 μg dose

[0577]

[0578]

[0579] Table 34

[0580] Tolerability score in mice at 700 μg dose

[0581]

[0582] Table 35

[0583] Tolerability score in mice at 700 μg dose

[0584]

[0585]

[0586] Table 36

[0587] Tolerability score in mice at 700 μg dose

[0588]

[0589]

[0590] Table 37

[0591] Tolerability score in mice at 700 μg dose

[0592] Compound No. 3 hours FOB PBS 0.00 1263826 0.00

[0593] Table 38

[0594] Tolerability score in mice at 700 μg dose

[0595] Compound No. 3 hours FOB PBS 0.00 387954 4.00 1287048 0.00 1287049 0.00 1287050 2.00 1287051 3.25 1287052 3.50 1287053 2.75 1287054 2.00 1287055 3.25 1287056 4.00 1287057 3.00 1287058 4.00 1287059 4.00 1287060 4.00 1287061 4.00 1287062 3.50

[0596] Table 39

[0597] Tolerability score in mice at 700 μg dose

[0598] Compound No. 3 hours FOB PBS 0.00 1287106 3.50 1287107 4.00 1287108 3.75 1287109 3.25 1287110 3.00 1287111 4.75 1287112 4.00 1287113 3.50 1287114 3.25 1287115 3.50 1287116 4.00 1287117 4.25 1287118 3.00 1287119 3.50 1287120 3.75 1287121 2.75

[0599] Table 40

[0600] Tolerability score in mice at 700 μg dose

[0601] Compound No. 3 hours FOB PBS 0.00 1287063 0.00 1287064 0.00 1287065 1.00 1287066 3.75 1287067 1.00 1287068 2.50 1287069 2.25 1287071 1.00 1287072 3.00 1287073 3.75 1287074 1.75 1287075 3.50 1287076 2.00

[0602] Table 41

[0603] Tolerability score in mice at 700 μg dose

[0604] Compound No. 3 hours FOB PBS 0.00 1287070 2.00 1287701 2.50 1287702 3.75 1287703 3.75 1287705 4.00 1287706 4.00 1287707 4.00 1287709 4.75 1287710 4.00 1287711 4.75 1287712 4.00 1287713 4.00 1287714 3.50 1287715 4.00 1287716 4.00 1287717 3.25

[0605] Table 42

[0606] Tolerability score in mice at 700 μg dose

[0607]

[0608]

[0609] Table 43

[0610] Tolerability score in mice at 700 μg dose

[0611] Compound No. 3 hours FOB PBS 0.00 1287122 0.00 1287123 0.00 1287124 3.50 1287125 3.00 1287126 3.00 1287127 0.00 1287128 0.00 1287129 4.00 1287130 2.75 1287131 2.50 1287132 2.75 1287133 3.25 1287704 3.50 1287708 3.50

[0612] Table 44

[0613] Tolerability score in mice at 700 μg dose

[0614]

[0615]

[0616] Table 45

[0617] Tolerability score in mice at 700 μg dose

[0618] Compound No. 3 hours FOB PBS 0.00 1318757 4.00 1318758 4.25 1318759 3.75 1318760 3.75 1318761 4.00 1318762 4.00 1318763 4.00 1318764 3.75 1318766 3.75 1318768 4.00 1318769 4.00

[0619] Table 46

[0620] Tolerability score in mice at 700 μg dose

[0621]

[0622]

[0623] Table 47

[0624] Tolerability score in mice at 700 μg dose

[0625] Compound No. 3 hours FOB PBS 0.00 1332247 1.75 1332248 0.25 1332249 0.00 1332250 3.75 1332251 0.00 1332252 3.00 1332263 2.00 1332265 1.50 1332266 1.00 1332267 3.75 1332268 2.75 1332269 1.25 1332270 2.25 1332271 2.50 1333508 0.00

[0626] Table 48

[0627] Tolerability score in mice at 700 μg dose

[0628] Compound No. 3 hours FOB PBS 0.00 1332255 1.00 1332256 2.00 1332257 1.25 1332258 1.25 1332259 2.25 1332260 2.25 1332261 2.50 1332262 2.00

[0629] Table 49

[0630] Tolerability score in mice at 700 μg dose

[0631] Compound No. 3 hours FOB PBS 0.00 1358996 0.00 1364777 2.00 1364778 3.00 1364779 3.50 1364780 3.50 1364781 5.25 1364782 2.50 1364783 3.50 1364784 3.50

[0632] Example 7: Tolerability of modified oligonucleotides complementary to human SMN2 in rats, long-term evaluation

[0633] In a separate study conducted under the same conditions, the selected modified oligonucleotides described above were tested in Sprague Dawley rats to assess long-term tolerance. Comparative compound numbers 396442 and 819735 were also tested in this assay. Sprague Dawley rats each received a single intrathecal (IT) delivery dose of 3 mg oligonucleotide or PBS. Starting 1 week after treatment, each animal was weighed and adverse events were evaluated weekly by trained observers. Adverse events are defined as atypical neurological dysfunction in PBS-treated control animals, including but not limited to: abnormal limb opening, abnormal gait, tremor, abnormal breathing, paralysis and spasm. The onset of adverse events is defined as the first week of dysfunction recorded after administration. If no adverse events are achieved, there is no onset (-). The onset of adverse events is usually associated with growth disorders as defined by lack of weight gain / maintenance, similar to PBS-treated animals. Similar tolerability assessments are described in Oestergaard et al., Nucleic Acids Res., Nov. 2013, 41(21), 9634-9650 and Southwell et al., Mol Ther., Dec. 2014, 22(12), 2093-2106.

[0634] At the end of the study, rats were killed and tissues were collected. Histopathological examination was performed on cerebellar sections using calbindin staining. As indicated in the following table, Purkinje cell loss was observed in cerebellar sections stained with calbindin. Cerebellum and spinal cord were also evaluated using antibodies specific for modified oligonucleotides. Animals showing no oligonucleotide uptake were excluded from histopathological analysis. For animals killed in advance due to adverse events, histology was not completed. In addition, cortical GFAP, a marker of astrogliosis (astrogliosis) was measured using RT-PCR (Abdelhak et al., Scientific Reports, 2018, 8, 14798), and the average increase of >2 times is described below.

[0635] Table 50

[0636] Long-term tolerability in rats at a 3 mg dose

[0637]

[0638] Example 8: Tolerability and pharmacokinetics of modified oligonucleotides in non-human primates, single or repeated dosing

[0639] Cynomolgus monkeys were treated with modified oligonucleotides to determine the local and systemic tolerability and pharmacokinetics of the modified oligonucleotides. Each group received artificial CSF or a single intrathecal lumbar bolus dose injection (IT) of the modified oligonucleotide, or for repeated dosing groups, the first day of the study was an IT bolus dose, followed by an IT bolus dose at a later time point. Tissues were collected 1 week after the last injection.

[0640] In a single-dose study, a single dose of modified oligonucleotide was administered to monkeys and tolerability was assessed. Representative doses for single-dose studies in adult cynomolgus monkeys include 1 mg, 3 mg, 7 mg, and 35 mg.

[0641] In the repeat dosing studies, monkeys were administered an IT bolus dose on study day 1, followed by weekly (e.g., days 8, 15, and 22 of a four-week study) or monthly (e.g., days 29, 57, and 84 of a 13-week study) IT bolus doses. Representative doses for repeat dose studies in adult cynomolgus monkey studies include 1 mg, 3 mg, 7 mg, and 35 mg.

[0642] The assessment of tolerance is based on clinical observations, body weight, food consumption, physical and neurological examinations (including sensory motor reflexes, brain reflexes and spinal reflexes), coagulation, hematology, clinical chemistry (blood and cerebrospinal fluid (CSF)), cell counts and anatomical pathology evaluation. A complete necropsy is performed and any macroscopic abnormalities are recorded. Organs are weighed and microscopically examined. Blood is collected for supplementary analyses. In addition, blood, CSF and tissues (at necropsy) are collected for toxicokinetic evaluation.

[0643] The tolerance of modified oligonucleotides in brain and spinal cord tissues was analyzed by measuring the Aif1 and Gfap levels of cynomolgus monkeys treated with modified oligonucleotides or controls. Brain and spinal cord samples were collected and flash frozen in liquid nitrogen and stored frozen (-60°C to -90°C). At the time of sampling, samples were collected from frozen tissues using a 2mm biopsy punch for RNA analysis. Punches were performed in multiple brain and spinal cord regions.

[0644] Example 9: Phase Ia human clinical trial with compound No. 1263789, 1287717, 1287745 or 1358996

[0645] The safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of modified oligonucleotides complementary to human SMN2 were evaluated in a clinical trial setting. Single and / or multiple doses of the modified oligonucleotides were evaluated in patients with confirmed SMA (e.g., SMA Type I, SMA Type II, SMA Type III, or SMA Type IV).

[0646] Patient safety was closely monitored during the study. Safety and tolerability evaluations included: physical examination and standard neurological assessments (including baseline), vital signs (HR, BP, postural changes, weight), ECG, AEs and concomitant medications, Columbia Suicide Severity Rating Scale (C-SSRS), CSF safety labs (cell count, protein, glucose), plasma lab tests (clinical chemistry, hematology), and urinalysis.

[0647] Select efficacy assessments that are appropriate for age and type and include, for example: the Hammersmith Motor Function Scale-Expanded (HFMSE), a reliable and validated tool for assessing motor function in children with SMA; the Pediatric Quality of Life Questionnaire (PedsQL TM ) Measurement 4.0 Common Applicable Core Scale; Children's Quality of Life Questionnaire 3.0 Neuromuscular Module; Compound Muscle Action Potential (CMAP); Motor Unit Number Estimation (MUNE); Upper Limb Module (ULM) and 6-Minute Walk Test (6MWT) (Darras et al., Neurology, 2019, 92: e2492-e2506).

[0648] Example 10: Design of modified oligonucleotides complementary to human SMN2 nucleic acid

[0649] Modified oligonucleotides complementary to human SMN2 nucleic acid were designed and synthesized as indicated in the table below.

[0650] Each modified oligonucleotide listed in the following tables is 100% complementary to SEQ ID NO: 1 (GENBANK accession number NT_006713.14, truncated from nucleosides 19939708 to 19967777). "Starting site" indicates the most 5'-nucleoside complementary to the modified oligonucleotide in the target nucleic acid sequence. "Stop site" indicates the most 3'-nucleoside complementary to the modified oligonucleotide in the target nucleic acid sequence.

[0651] The length of the modified oligonucleotide in the following table is 18 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, wherein each 'e' represents a 2'-MOE sugar moiety, and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a thiophosphate internucleoside linkage, a phosphodiester internucleoside linkage, a methoxypropylphosphonate internucleoside linkage, or a methylsulfonylphosphoamidate (MsP) internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, wherein each 's' represents a thiophosphate internucleoside linkage, each 'o' represents a phosphodiester internucleoside linkage, each 'x' represents a methoxypropylphosphonate internucleoside linkage, and each 'z' represents a methylsulfonylphosphoamidate (MsP) internucleoside linkage. Each cytosine is 5-methylcytosine. Modified oligonucleotide 449320 has been previously described in WO 2015 / 161170 A2.

[0652] Table 51

[0653] MOE and NMA modified oligonucleotides with mixed PO / PS, PO / MsP, uniform MsP or PS / MOP internucleoside linkages

[0654]

[0655] The modified oligonucleotides in the following table all consist of the following sequence (5' to 3'): TCACTTTCATAATGCTGG (SEQ ID NO: 23). Each modified oligonucleotide listed in the following tables is 100% complementary to SEQ ID NO: 1 (described herein above). The "starting site" indicates the 5'-nucleoside most complementary to the modified oligonucleotide in the target nucleic acid sequence. The "termination site" indicates the 3'-nucleoside most complementary to the modified oligonucleotide in the target nucleic acid sequence.

[0656] The length of the modified oligonucleotide in the following table is 18 nucleosides. Each nucleoside comprises a 2'-MOE sugar moiety or a 2'-NMA sugar moiety. The sugar motif of each modified oligonucleotide is provided in the sugar motif column, wherein each 'e' represents a 2'-MOE sugar moiety, and each 'n' represents a 2'-NMA sugar moiety. Each internucleoside linkage is a thiophosphate internucleoside linkage, a phosphodiester internucleoside linkage, or a methylsulfonylphosphoramidate (MsP) internucleoside linkage. The internucleoside linkage motif of each modified oligonucleotide is provided in the internucleoside linkage motif column, wherein each 's' represents a thiophosphate internucleoside linkage, each 'o' represents a phosphodiester internucleoside linkage, and each 'z' represents a methylsulfonylphosphoramidate (MsP) internucleoside linkage. Each cytosine is 5-methylcytosine. The modified oligonucleotides in the table below are conjugated with a 6-palmitamidohexyl phosphate conjugate group attached to the 5'-OH of the oligonucleotide. The structure of the conjugate group is:

[0657]

[0658] Table 52

[0659] 6-Palmitamidohexylphosphate-conjugated MOE and NMA-modified oligonucleotides with mixed PO / PS, PO / MsP or uniform MsP internucleoside linkages

[0660]

[0661] The modified oligonucleotides in the following table all consist of the following sequence (5' to 3'): TCACTTTCATAATGCTGG (SEQ ID NO: 23), the starting site is 27062 on SEQ ID No: 1 (described herein above) and the ending site is 27079, wherein the "starting site" indicates the 5'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide, and wherein the "ending site" indicates the 3'-nucleoside in the target nucleic acid sequence that is complementary to the modified oligonucleotide.

[0662] The modified oligonucleotides in the following table are 18 nucleosides in length. The sugar and internucleoside linkage motifs of each modified oligonucleotide are provided in the sequence and chemical notation columns, wherein each subscript 'n' represents a 2'-NMA sugar moiety, each subscript '[DMA]' represents a 2'-O-(N,N-dimethyl)acetamide moiety, each subscript '[NEA]' represents a 2'-O-(N-ethyl)acetamide moiety, each subscript '[NPA]' represents a 2'-O-(N-propyl)acetamide moiety, each subscript '[NcPA]' represents a 2'O-(N-cyclopropyl)acetamide moiety, each subscript '[McPA]' represents a 2'-O-(N-cyclopropylmethyl)acetamide moiety, and each subscript 's' represents a phosphorothioate internucleoside linkage. Each cytosine is 5-methylcytosine, wherein in the cytosine residue ( m C) before the subscript 'm' represents 5-methylcytosine. The structure of each sugar shown in the following table is:

[0663]

[0664] Table 53

[0665] NMA and NMA analog modified oligonucleotides with uniform PS internucleoside linkages

[0666]

[0667] Example 11: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, single dose (35 μg)

[0668] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2.

[0669] deal with

[0670] Transgenic mice were divided into groups of 4 mice per group. Each mouse received a single ICV bolus of the modified oligonucleotide at the dose indicated in the table below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the coronary brain and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as the fold change of RNA levels normalized to total SMN2 levels relative to the PBS control. Exon inclusion (Exon 7) was calculated using a nonlinear regression 4-parameter dose-response curve [Y = bottom + (top - bottom) / (1 + (10^logEC50 / X)^Hill slope)] in GraphPad Prism 7. + ) 50 .

[0671] RNA analysis

[0672] 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 exon 7 ( + The amount of SMN2 RNA was determined using the primer probe set hSMN2_Sumner68_PPS50481, which does not include exon 7 (exon 7 - ) of SMN2 RNA. Total SMN2 RNA levels were measured using primer probe set hSMN2_LTS00935. Results are presented as fold change in RNA levels normalized to total SMN2 levels relative to PBS control.

[0673] Table 54

[0674] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0675]

[0676] Table 55

[0677] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0678]

[0679] Indicates that fewer than four samples are available

[0680] Example 12: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, single dose (15 μg)

[0681] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2.

[0682] deal with

[0683] Transgenic mice were divided into groups of 4 mice per group. Each mouse received a single ICV bolus of the modified oligonucleotide at the dose indicated in the table below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the coronary brain and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as the fold change of RNA levels normalized to total SMN2 levels relative to the PBS control. Exon inclusion (Exon 7) was calculated using a nonlinear regression 4-parameter dose-response curve [Y = bottom + (top - bottom) / (1 + (10^logEC50 / X)^Hill slope)] in GraphPad Prism 7. + ) 50 .

[0684] RNA analysis

[0685] 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 exon 7 ( + The amount of SMN2 RNA was determined using the primer probe set hSMN2_Sumner68_PPS50481, which does not include exon 7 (exon 7 - ) of SMN2 RNA. Total SMN2 RNA levels were measured using primer probe set hSMN2_LTS00935. Results are presented as fold change in RNA levels normalized to total SMN2 levels relative to PBS control.

[0686] Table 56

[0687] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0688]

[0689]

[0690] Example 13: Activity of modified oligonucleotides complementary to human SMN2 in transgenic mice, multiple doses

[0691] The activity of the selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2.

[0692] deal with

[0693] Transgenic mice were divided into groups of 4 mice per group. Each mouse received a single ICV bolus of the modified oligonucleotide at multiple doses as indicated in the table below. One group of 4 mice received PBS as a negative control. Two weeks after treatment, the mice were sacrificed and RNA was extracted from the coronary brain and spinal cord for real-time qPCR analysis of SMN2 RNA. The results are presented as the fold change of RNA levels normalized to total SMN2 levels relative to the PBS control. Exon inclusion (Exon 7) was calculated using a nonlinear regression 4-parameter dose-response curve [Y = bottom + (top - bottom) / (1 + (10^logEC50 / X)^Hill slope)] in GraphPad Prism 7. + ) 50 .

[0694] RNA analysis

[0695] 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 exon 7 ( + The amount of SMN2 RNA was determined using the primer probe set hSMN2_Sumner68_PPS50481, which does not include exon 7 (exon 7 - ) of SMN2 RNA. Total SMN2 RNA levels were measured using primer probe set hSMN2_LTS00935. Results are presented as fold change in RNA levels normalized to total SMN2 levels relative to PBS control.

[0696] Table 57

[0697] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0698]

[0699] Table 58

[0700] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0701]

[0702]

[0703] Indicates that fewer than four samples are available

[0704] Table 59

[0705] Effects of modified oligonucleotides on human SMN2 RNA splicing in heterozygous transgenic mice

[0706]

[0707] Example 15: Tolerance of modified oligonucleotides complementary to SMN2 in wild-type mice

[0708] The modified oligonucleotides described above were tested in wild-type female C57 / Bl6 mice to assess the tolerance of the oligonucleotides. Wild-type female C57 / Bl6 mice each received a single ICV dose of 700 μg of the modified oligonucleotides listed in the table below. Each treatment group consisted of 4 mice. A group of 4 mice received PBS as a negative control for each experiment (identified in the following separate table). Three hours after injection, mice were evaluated according to seven different criteria. The criteria were (1) mice were smart, alert, and reactive; (2) mice stood or arched their backs without stimulation; (3) mice showed any movement without stimulation; (4) mice showed forward movement after they were lifted; (5) mice showed any movement after they were lifted; (6) mice responded to tail pinching; (7) even breathing. For each of the 7 criteria, if the mouse met the criteria, the mouse was given a sub-score of 0, and if it did not meet the criteria, a sub-score of 1 (Functional Observation Experiment Combined Score or FOB) was given. After evaluating all 7 criteria, the scores for each mouse were summed and averaged within each treatment group. The results are presented in the table below.

[0709] Table 60

[0710] Tolerability score in mice at 700 μg dose

[0711] Compound No. 3 hours FOB PBS 0.00 1287723 2.00 1287724 1.00 1287727 2.00 Sequence Listing <110> Ionis Pharmaceuticals, Inc. <120> Compounds and methods for modulating SMN2 <130> BIOL0367WO <150> 62 / 983,545 <151> 2020-02-28 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 28070 <212> DNA <213> Homo sapiens <400> 1 ccacaaatgt gggagggcga taaccactcg tagaaagcgt gagaagttac tacaagcggt 60 cctcccggcc accgtactgt tccgctccca gaagccccgg gcggcggaag tcgtcactct 120 taagaaggga cggggcccca cgctgcgcac ccgcgggttt gctatggcga tgagcagcgg 180 cggcagtggt ggcggcgtcc cggagcagga ggattccgtg ctgttccggc gcggcacagg 240 ccaggtgagg tcgcagccag tgcagtctcc ctattagcgc tctcagcacc cttcttccgg 300 cccaactctc cttccgcagc ctcgggacag catcaagtcg atccgctcac tggagttgtg 360 gtccgcgttt ttctacgtct tttcccactc cgttccctgc gaaccacatc cgcaagctcc 420 ttcctcgagc agtttgggct ccttgatagc gttgagtgga ggccctgccg cgacttggca 480 gtagcttatt ttgttcactc ctctctggct ggtgtggggg aggtgggggc attaggccag 540 ggtgaagcag gggaaccact taggagtctg ttaagatgat ctgaacttca gaacaagatg 600 ttattaacag agtgaaagta tttggattct gggtatattt tgaaatcgga ggcaacaggt 660 ttttcagata gattcgata cggaggttat cctgaatagt tgaaaagata aagttgcctt ttgctgaggt gggaaaga agattgccag taggcaggt ttctcaggag ttcagtcttg 780 ggcatagcat ggtaggggtg aatttggctg gagtgagttg gagagtagga gaagagaaat 840 ccaaggcaac atttgaccag cctgggcaac atagtgtgac tccgagtctg caaaaattag acgggtgttg tggtgcgcgt ctgtggtctc agctacctgg aaggttcagg ccttggaagg 960 ctcagggagg tggaggctgc agtgatctgt gattgcgcct ctgcactcca gcctgggcga cagagccaga ccctgtctta aaacaaaata aacggccggg cgcggtggct caagcctgta atcccagcac tttgggaggc cgaggcggcc ggatcacaag gtcaggagat cgagaccatc ctggctaaca cggtgaacc ccgtctctac aaaaattagc cggggcgtggt gacgggcgcc tgtagtccca gctactcggg aggctgaggc aggagaatgt catgaagccg ggaggcggag cttgcagtga gccgagatcg cgccactgca ctccagcctg ggcgatagg 1320 caagactccg tctcaata father father father father catcggtagg catatttcaa ggaattctat ttaaaaaaaa ttttttaga gandaagttcg ctctctgtgg 1440 cccaggctgg agtacagtgg catgatccta gcccatggca gcgttgatct cttggcctca 1500 agcgaccctc ctttggagtc gctggggccta aaggagtgag ccaccacgaa atttattat 1560 aaatggagggg tagagaattt gggcaataa tggaggggga agtgagtta gaggaattttt 1620 aattatgtgt gtgtggtttt aaagagggg ggtcttgctc tgttgcccag gctgctgggg 1680 tgccagtgc gcaatcatga atcactacag ccttggactc ctggcctca gctatcctcc 1740 cacctctgcc tcccaagta ctgggattac tagtgtgagc cactgcacta agataggagc 1800 aacatgtttc agcatgtttg tgggttgata ggaagatga gatgggaaa gttgatgtcg 1860 gaagaagac aatggctaga gcaatgtcct agagtaggta agaaggatg gattggct 1920 ttgttggaaa cattagcggt tcttttgtg acagctatat agttaacaca tctatgatac 1980 gtgaatgggc agataggatg gcaggagatt tgaaagttc tcttgattct tactgttctc 2040 tagtgaag aagcaggtt atcagctaga agctgggatg ggagagaa gagaagatgg 2100 gaagtagata gttctttaga agagtgggca agggttggac tagggatt taggtggaat 2160 attgctaggc aacataaga gcctacttga gattcgtggt catgagttga aggagaccag 2220 acagcagat tgtgtatgag ggcaccaca gagtaaatgg agagttgaa ttaatgcagt 2280 tgtgatttta cacgtggat atgagaagt gagggggaga agtacaagg agttctctta 2340 atgattgacc atgaattta agctggctaa gaaggaagt gagaggccgg gcgcggtggc 2400 tcacgcctgt atcccagca ctttgggaga ctgaggtggg tggattact gaggtcagga 2460 gtttgagacc aacctggccg atatggcgaa acccactc tataaaaat acagaaaaat 2520 tagccgggaa tggtggcagg tgcctgtaat cccagctact tagccggctg tggcaggt 2580 atcccttgga cccaggaggt ggaggttgca gtgagccgag atcacgccac tgtactccag 2640 cctggacgat atagtgagac ttcacctca aaaaaaaaa aaagaaagga agtgaggatt 2700 ttaagaccct gagacacagt ttaaaagtg ggaggatcgg ccggcgctg tggctgacac 2760 ctgtaatccc agcactttgg gaggccgagt tggcagatc acaggtcag gagttcgaga 2820 ccagcctggc caatatggtg aaaccttgtc tctactaaaa atacaaaaat tagccgggca 2880 tggtgtcacg tgtctataat cccagctact cgggaggctg aggcagaaaa attgcttgaa 2940 cctgggaggc agaggttgca gacagctgag atcactccat tgcactccag cctgggcaac 3000 aagagcaaaa ctttgtcttt aaaaaaaaa aaaaaaaaag aatacaaaaa ttagccgggc 3060 gtggtggcgc gtgcctataa tcccagctac ttgggaggct gaggcaggag aatcagttga 3120 acacgggagg cgaggtttgc agtgagccga gattgcgcca ctgcactcca gcctgggcga 3180 cagagcagga ctcctcttgg aaaaaaaaa ttagctgggc atggtggcag gtgcctgtag 3240 tctcagctac tagggaggct gaggcaggaa aatcacttga acccgggatg tggagtttgc 3300 agtgacccga gatcgtgcca ctgtactcca tctgggcga caaaatgaga ctctgcctca 3360 aaaaaaaaa aaaaaaaaag tgggaggatc aatgtactgc cagtcctaat gaagtggaat 3420 gattgtcccc atcaaatcac tagtaggagt aagttgcaga gcctagaagg tgatggttaa 3480 gagagtggga ttcttgaaac tgcatttatg gagaggttgt ggttattggt tataataaat 3540 aaatacagtt gaagtgagtg agtaggctgag attggggat gtatcagttc attcttacac 3600 tgctacaaag acatacctga gaccaggtat ttataaagat aagaggttta atcagctcac 3660 agttctgctg cctgtacagg cttctcttgt ggaggcctaa ggaacttac agtcatggtg 3720 gaaggtgaag gggaaacaag cacagtcttc acatggccag caggagagag agaagggg 3780 3840 ttggggatgg tgctaaatca ttagaaatca cccccatgat ccagtcgcct cctaccatgc 3900 ccacctccaa cactggggat caaattcag catgagattt gggtaggaac acagagctgc 3960 accacatcag aggatgtaca agattgtggt ggagagggt ttagagacct gcaaatatag 4020 ggtaattgaa gggatcatct acatggatat ttaaatcacc aaaaattatg acaggagtag 4080 tgttggagag agaactgcga tgtaaacatt aaagaatgag gaagagtgac tcggtaggct 4140 gtaggtgact gcataggaa aggataatag actgtgagtc tggtgacaag attttcctt 4200 tttctttttt tcccccccc cgagacaggg cctctttttg ttgcccaggt gggagtgcag 4260 tggcgcgatc acggctcact acacctcct cccagctca agggattctc acggtcagc 4320 ctctcaagta gctggaacta caggtgctga ccaccatgcc tggctacttt tgtcaggat 4380 tttcaggct gggaatttg agagggaat ggaggagaat atctgaag tgcaagtaag 4440 gagcaggga gatttctttttttttttttttttttttgagtcggag tctggctcag 4500 tcgcccaggc tggagtgcag tggcgagatc tccgctcact gcaagctccg cctcccgtgt 4560 tcacgccatt ctcctccttc agcctcccga gtagctggga ctacaggcgc ccgccaccac 4620 gcccagctaa ttgtttttt gtatttttag tagagacggg gttcaccgt gttagccagg 4680 atggtctcaa tctcctgact ttgtgatccg cccaccccgg cctcccaag cgcttgggat 4740 tacaggcgtg agccaccgcg ccagccagag cagggaagat ttctcccca catctccagt 4800 aggtacagtg atatgaagtg tgtggaggag aaagaggaa acatctca tttgagatgg 4860 ctgcgaaagg aaaggcatc ctcagggagc tagattttac ttagagcaag aaatgaaggg 4920 atgattcaga gttaaaga gtggatttta tgaattactc aagggagcac agtggaagtt 4980 tcaggaagtg gtaggagaag gtagaagatg gcagggtgtt gggaataatt tgagaaatct 5040 gagctactgg aaatgactga gaatcagata taaaggcagt cctggtggtc cgttctggct 5100 gccgttgctg tgtaacgaat ctgccaaaac ttagtggctt gaaacaacaa agaacatttt 5160 attatctctc attgtttctg tgggttagga atttgtgaga gccgtgctgg gcagttttcg 5220 tgcggctgtc tcgtggttgc acctacatag ttgctagagc tacagtagct ggggactgag 5280 cagctaggga ttggcaggct atctcttttt ttcatgtagt ctcatgaaga tttctttatg 5340 tggtttcaat gtgtgggctg gtttggattt ccttatagca tggtggcctc agttggattg 5400 ctgttttgtg atccttttca tccctccttg tcctgtcccc agacaaccac tgatctactt 5460 tctgtcacca tagattagcc tgcattttta agaattttta taaacgtgga atgatagagt 5520 accttttttg tcacgtttct tttatttatc atagctattt tgattttcat ccattttatt 5580 gctgagtagt atcccattgc atgtatatac tatactgtat tcattcgctt gcttgtgaac 5640 atttgggctt tttccagttt gggactgtta acaagtagag ccactatgaa tattagtgta 5700 taagacttca tatagccaag gctggcagat cgcttgagcc caggagtttg agaccagcct 5760 gggaaacatg gtgaaacctc tatttttatt ttaaaatcaa aaattaaaaa ttttctataa 5820 aaaattttaa agaagacttt gtatagacat acgctttcat ttttcttgag tgaatactta 5880 ggtctcaggg tagatgtatt ttaagtcttt aaggagctgt caaactcttc ctcaaagtgg 5940 tggttgtacc atgttacttt ttaatataac agagattaat tgagcaaaga aaaattcaaa 6000 agttggacag cccccacaac taataggtt cagaacagct cccccatttt gcattttgac 6060 cagcaatgta tgaaagttcc atttgctcag tgtccctgca aacacctggt atggtcagtc 6120 ttttaattt taggcattat ataagatata gtggcttctt gtgattttaa ttagcatttc 6180 ctaatgacca gtgctgctgt tgatcatttc atgagtgtat ttgccatccg tatatctttt 6240 ttggtgaagt gtctattcaa atcatttggg tttttttttt ttttgttttt ttttttgga 6300 gacagtgtct cactctgtca cccaggctgt tgtgcagtgg tgcaatcaca cagcctactg 6360 cagcctccac ctcctgcgct cagctttctt gtctcagcct tctgagtagc tgaaattacg 6420 agcacacgcc acaatgcctg gctaattttt taaaatttg tagaaacaag gtctcattat gttgcctggg cttgtcgtga actcctgggc tcaagcaatc ttcctgcctc agcctcccaa 6540 agttgggat tgcaagtatg agccactgca cccggccaac ttacccatct tttaattgaa tttttttgtt gttgaggttt gagagttctt catgtttgct gggtacaata tctttatcag ataggtaact tgcatgtatt ttctcccggt ttacactttg gtttttcatt ttgttaacaa cgtcttttta agaacagaaa atcttaattt tgctgaaatc taatttttca gttttttctt tgatggtttt gagagaggag gtaaaaaaag actaggtaag ccgatagtta gacagagtcc tcggtagaac ttcccttcta acaaaaagca gcccaagaaa tcacttctct tctaacaagg agcagcctgg aagatcgggc tgtaaacatg tataaggag cagctctggc acagaggggg agcttcctgg gtaatcagca agcttcacat acgtaaggtg ggtatgtgaa gtaaacacag tatgtgaagt aaacacagtg gaccttagta catactcaga tagcttgct ggaagcttgc atgttgtgag ttgttggggt tgcctgcagc tgcacggaga gaaaggggta cctggggcca 7140 ggcatgtcca ccatggtggc tccacctccc cttatttagc acatgcacaa taggaaagag 7200 ataagcaatg tggagtagct caggccaagg acctgcctgc ataataaaag gttggggtgg 7260 gggatgccag agattcacgc tctgtgcaga tggcaacacc tggtcctaac tggttttttg 7320 ctccctatgt gtagataagc taccccttc ccattagctc atttataaaa atgcttgcat 7380 ttcactgtgg aatgggaact cttttcagga cctctctctg caggagagag ctagtctctt 7440 tctttgcct attaaacttc tgctctagcc tcacaccctt ggtgtgtcag cgtccttgat 7500 ttcctcagcg tgagaccaag aacctcgggt gccaccccag gcaacaaggc catttcagtt 7560 tgttctttg ttataggcaa tccatgatca cagatttttc tctcttttt ttttttacac 7620 agttagagt tttagtttta cacttaggtc tgtaatccat tttgtattaa ttcttatatg 7680 tggctcagtg taggtggaaa tttggtttgt ttttgcataa ggatttccaa tagttttacc 7740 accatttctt gaaactacta tgctttctct attaaaccac atttgtaact ttagttaaaa 7800 tcagtcacat atatcacagg gctatttctg actctcaatt ctgttacatt gtctattagt 7860 gtatattgat gtcagtacta cacttttaat tactattgct tcagggtatg tcttgtaaac 7920 caaaaataaa attataggcc ccccccgcc ctgcacaacc aactgaatgg acccatcctc 7980 tcagccaagg gcattccaaa attaacctga aaaactagtt caagccatga tgggaagggg 8040 gagttggaca tgtctcatca caccctacta ccttttggaa ttactgatag aacagactct 8100 taaagtctga aaagaaacat ttacaaccta ccctctctga agcctgctac ctgggagctt 8160 catctgcatg ataaaacctt ggtctccaca acccctatg gtaacccaaa cattcctttc 8220 tgttgataat aactctttca actagttgcc aattagaaaa tctttaaatc ttcctatgac 8280 ctagaaacct ccctaccccc actttgagtt gtcctgcctt tcctgacaga actcatgtac 8340 atcttacata tattgattga tgcctcatgt ctccctaaaa tgtataaaac aaagctgtac 8400 cccaccacct tggggacatg tcatcaggac ctcctgtggc tgtgtcatag gagcgtcttt 8460 aactttggca aaataaactt tctaaattga ttgaaacctg tcttagctac ttctggttta 8520 cagtcttaaa gttagataat gtaaattgtc cagctttggt ttatttttgt ccttagtagt 8580 tccatataaa ttttagaatc agcttttcaa tttaatacac tactttcctc ttagatccac 8640 aattaaatat atttgatgct aacaattctg ttttatgttt ttcgtttttt ttttttgaga 8700 caagagtttc gctcttgttg cccaggctgg agtgcagtgg cgcgatcttg gctcaccaca 8760 acctccacct cccaggttca agcaattctt ctgcctcagc ctcccgagta gctgggatta 8820 caggcatgcg ccaccacgcc cggctaattt tgtattttta gtagagacgg ggtttcacca 8880 tgttgatcag gctggtcttg aactcctgac ctcaggtgat ccacccacct cggcctccca 8940 aagtgttggg attacaggcg tgaaccacca tgcctggcca gttctgttat tttaaaacc 9000 caagtttccc tggtcatatc ttggttggat gaagcgtatt ttcaatagat taccctggaa 9060 aggctagtga gtacggtatt cttctacatt ttagactttt cttagtcttg ctacttcaag 9120 gacagctagg ctgcatataa aattcttggc tcatactttt tccccataaa tttctatgag 9180 aaagtctaat gataactgat tttctttatt ttgtaactta gtctttttgc ttagaggctc 9240 tctgaggatg ggaggggtt cttcctccca tccctaggaa ttttcttttt ttttaaattc 9300 ctaatcacta gaccaccagg aagattgttt gttttgtttt gtttttattc ttcagggacc 9360 ccatttatac atacgttaaa taaatactgt ttgccaatgt atcaaccatt ttgcttctta 9420 tttatttttg ttcctttggt tctttttcat ggctttgctt tggtgctcct tagattttca 9480 gtcagatgta tttgtccttg ggtaccttgt aatcagtatt accttttctt ctgtcgcttt 9540 gttttctgtt cgttttgaaa ttacttgttt cctggtctgg caataacagt tgagatatga 9600 ggagtttgag ctgccatctg tctatgtatc ttgctttaag actgcactct tctattgata 9660 tcactggcct tgattttgtg atttctttat ttcttcagga ccacccttca ttttctactg 9720 tttgcttcct ttttttttga gatggagtct cactctgtca ctcaggctgg agtgcagtga 9780 tcttggctca ttgcaacctc tgcctcccgg gttccagcaa ttctcctgcc tcagcctccc 9840 aagtatctgg gactacaggt gtgcaccacc atgcccggct aagttttgta tttttaatag 9900 agacggggtt ttgccacatt ggcaggctgg tctcaaactc ctgatgtcaa gtgatccacc 9960 caccccaccc acctctgcat cccaaagtgc tgggattaca ggaatgagct gccgtgccca 10020 gcctcccccc tacccccctt ttttctttc gagacagaga ttataggtgt gagccactgg 10080 acccagcctg ttttattcc ttttaccaaa tctccaagga atactctccc ttccaagtgc 10140 gaatgtaacc ttaagtcagt taacctcttt gtgattactt ttcttatctg caaagtgact 10200 taatgatctt aagtacttttt ttttttgag acagggtctc actgtcaccc tggctggagt 10260 gcagtggcac gatctctgat ctccactcac tgcaatctcc tcttccctgg ttcaagcggc 10320 cctcccacct tagccttctg ggtagctggg actacagatg tgaaccacca cgcccagcta 10380 atttttgtac ttttttgtaga gatggggttt tgccatgttg cccaggctgg gattattaag 10440 tacttttat catacagcaa gattgacatt ttatattgga atacatttgt ctctatataa 10500 cggagattaa caggaaaatg acaagcctgg gtgcggtggc tcatgcctgt aatcccagca 10560 cttgggagg ctgaggtggg aggatcactt gaggtcagga gttcgagacc agttttgcca 10620 agatgatgaa agcccatgtc tactaaaaat acaaaaatta gcccagcttg atggtgggcg 10680 cctataatcc cagctatttg agagactgag gcaggagaat cacttgaacc tgggcagcag 10740 aggttgcagt gagccgagat catgccactg cactccagcc tgggtggcat agcgagactc 10800 ttgtctcaag agaaaacaaa acaaaacaaa aaaaaaacag gaaaatgaca aaaagtaata 10860 ttacaactca gtgaatttta taacaaactt ttttggaatt cattgactaa tactatacca 10920 aatccaaaat actctctagt ataccaaatc caactctacc ctatagtata aattggattc 10980 tatttggact tgtctcacta atccctcata cagtgtgttt tattttttat tgaagtaaaa 11040 aaatttgtca ttttaaccat ttttaagtat atagttcagt aatattaagt atgttcatgt 11100 tgttgcgcaa tagatcttcg gaagtttttc gtcttgcaac ctgaaactct acccattagc 11160 aaattcccat ttctccttac acttagccct tggtaatcat cattcttttt tttttttttt 11220 tgagatggag tttactctt gttgcccagg ctggagtgca atggtgcaat ctcgactcac 11280 cacaacctcc gcctcccagg ttcaagcaat tctacctcag cctcccgagt agctgggatt 11340 acagtcatgc accaccacgc ccggctaatt ttgtattttt agtagagaag gggtttctcc 11400 atgttgaggc tggtctcgaa ctcctgacct caggtgatct gcccacctcg gcctcccaaa 11460 gtgctgggat tacaggcgtg agccactgcg cctggcccat tctttctaat tctataaatt 11520 tgactactta gttaccttac ataaataaat tcttatagtt agtgttattt ttgcttccat 11580 gccttttttg ttgttgttca tgctcttact tggaatgcgt tctattttgt ctacctatgc 11640 acatcctgtt gggttttttt tttttttggg ggtttttttt gttttttttt gttttttttt 11700 cccagacaag gtctcaattt gttacccagg ctggagtgca gcggcgccat ctccactcac 11760 tgcatcctca acttcctggg cccaggtgat cctctcgcct cagcccctgc aggtagctgg 11820 gactataggc atgtgccacc atgcccagct aaatttggtt tttttgtttg tttgtttttg 11880 agacagagtc tcactctgtc acccaggctg gagtgcagtg gcacaatctc agctcactgc 11940 aatctctgcc gcccgggttc aagtgattct cctgcctcag cctcccaagc agctgggatt 12000 acaggtgact gccaccacgc cagctaagtt ttgtagtttt agtagagatg gggtttcacc 12060 ttgttggcca tgctggtctc gaactcctga cctcgtgatc tgcctgcttc tgcctcccaa 12120 agtgctggaa ttacaggcat gagccaccac gcccggccag aatttttgta tttttagtag 12180 acacaaggtt cttaccctgt tgcctaggct ggtctggaag tcctggactc aagcaattca 12240 cctgccttgg cctcccaaaa tgctgggatt acaagccacc atgcccggcc taaatcctgt 12300 tgttttgttt tgttttattt tgttttgttt tgttttgttt gttttttgag acagagtctc 12360 gctatgtctc tcaggctgta gtgcagtggc gcgatcttg ctcactgcca cctctgcctc 12420 ccaggttcaa gtgattctcc tgcctcagcc tcccaagtag ctgggattac aggcatgtgc 12480 tactatgtcc ggctaatttt tgtattttta gtagagacag ggtttcacca tgttggccag 12540 gctggtctcg aactcctgac ctcgtgatcc acccacctcg gccacccaaa gtgctgggat 12600 tacaggcgtg agtggttttt attcttagg ccggttttcct ccatatgatc ttgcagtaga 12660 cattaatttc tttcctttt attaaaata ctgtttgtat ttcacatttt gatgtttgtt 12720 aagatttgtt ttatattgtt ttttgttttg tcttgtgtga tagtcttaaa tccctagtta 12780 gataact ggagagtacc atgttctat atatctctca gtgacttgca cagtgctagc 12840 agatagtgct aaaaaattat ttattat tattatttg ttattgttgt tgttgttgtt 12900 agacagggtc ttcctctgtc acccaggcta gagggcaatg ggatgatcat agcttactgc 12960 agcctccaac aactgggctc atgttattct cctgcctcag cttcccaagt agctgggatt 13020 acaggcatga gccaccatgt ctggacaaaa atatttccag gtgcagtggc tcatgcctgt 13080 aattcccaca cttgggaggc cgagcgaggc tggaggatca cttgagccta ggagttcaag 13140 accagcttgg ctaagatggc gagaccccgt ccctacaa aattttaaaa actagccagg 13200 catggtggca tgcacctata ttcccacta ctcagtgggc tgaggtggga gggtcatttg 13260 aacacaggaa tttgagggagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaai agggagggagggaaaaaaaaaaaaaaaaaaaaaggggg 13380 aaattggtac caggaagca ggaagggaa atggaagtaaaaaatadadadad 13440 aatgaaaatt gttagtcac tattacaat ttgtacctt atatctgga aacattataa 13500 tttcaaaaga aaaaatattc tttggatcat aggttctgag gtcagaacag cattcccgta 13560 gtctagatga agtcaagttt tatctgatct taattgaaat aaatatagct ggccttgaac 13620 aaatctactc atggtatgtg gataggaatt aaattgtagg ggcattcact tgatggcatt 13680 cattcttaga acatttacct atgtctagct tttggagtaa agtcacataa cctctaacca 13740 ggtaagtttc ctgtggcttt atttaggatt ttaaatactc attttcagtg taattttgtt 13800 atgtgtggat taagatgact cttggtacta acatacattt tctgattaaa cctatctgaa 13860 catgagttgt ttttatttct taccctttcc agagcgatga ttctgacatt tgggatgata 13920 cagcactgat aaaagcatat gataaagctg tggcttcatt taaggtatga aatgcttgct 13980 tagtcgttt cttattttct cgttattcat ttggaaagga attgataaca tacgataaag 14040 tgttaaagta catgttattc agttttcatt ttgaagatta gatggtagta tgagttagtt 14100 aaatcaggtg atatcctcct ttagaagttg atagcctata tatgtcatcc tttgtggagg 14160 14220. ctggctgggt atggtggctc actcctgtaa tcccagcact ttgagaggct gaggcgggtg gatcacctga ggtcaggagt ttgagaccag cctggccaac atggtgaaac cccgtcttta ctaaaaatac aaaaattagc caagcatggt ggcacgtgcc tgtaatccca gctgcttggg acactgaggc aggagaattg cttgaacctg gggggcagag gttgcaatga ttgcaccact gcactccagc ctgggcgata gagtgagact 14460 ccatctcaga aaacgaacaa acaatgtatt ccttttagta tttttacatt gtatcaaact atggaagtcc tctaattgag attack aaaagacaat ctgaattata attack tttaacaagc atgtagtaaa attachment to the cattagtaca gcaattaata tttgtagcat gctgacagtg ctctgtgtgc gtttcatata ttaaattact ctaatcatcc 14760. caaatcctgt aagttgggta tcaattcaag tgttcctatt gggtaggaat atacagttct tttaggaaat gtagtatggt tctgtgtctc aaacaggaca cttacacagt tggccaacat catcaccttc tccattctct gagatgttta gtcttactga gcactaaata tgggtcatca atagtccaga ctaccttgag caaacaatag tccagactac cttgagcaaa cagagcatat 14940 actcatacag tgtataaaga gcaccaagca tacagatttc atgtctttct catagttact 15000 cttgtaacat gagctaaaga tcagacctct atgtcacctt tgtaactgat ttctagattt 15060 tttttttttt ttgagatggg gtcttgccct gtcacccagg ctggagtgta gtggcgtgat 15120 catgcctcat tggagccttc aactcatgag ctcaaacaat cctcctacct cagcttcctg 15180 agtagttggg accacaggtg tgtgccacca cacccagctc atttttgtat tctttgtaga 15240 gatgcagtct caccctgttg cccacgctgg cctggaactc ctgagctcaa aagatccctc 15300 cgccttgacc ttccaaagtg ctgggattac aagcatgaac cactgcaccc ggcctagatt 15360 tttaaatgtg ctttccagta tacactgaaa ctagaagtcg actaaagaat taccaagaga 15420 attctataaa atagagattg aaatggggct cgatgtggga tgggttggtg atattgcagg 15480 gagaagtaat ctgagtaaag gaggaaaaga actgatttgg gaaaacgata gttttagtag 15540 tgagtttgag tatgattga gttgattg aatttgatt aagttgaggt tgaatatga 15600 ttaagttgag gttgagttg aggtagat tagatgtga attgatcat tggatgtt 15660 agattgagaa aagtcacagc tggattata gcttcagaag tgtgttgca gagagttgca 15720 actaaagtaa tagaataga tggccttggc cggggcgcggt ggctcacgcc tgtaatccca 15780 gtactttggg aggctgaggc gagcaatca cgaggtcagg agttcagac cagcctggcc 15840 cacatggtga aaccccgtct ttattaaaaa tacaaaatt agctgtgcac agtggtgcac 15900 gcctgtaatc ccagctactc gggaggctga gaggagaa tcgcttgaac ctgggaggtg 15960 gaggttgcag tgagctgaga tcagtgtgac tgcactccag cccggtgaca gagtgagact 16020 ctgtgtaaaaaaaaaaaaaaat tggccgtaag caagtaag aggatggcca 16080 gctcttattg ggaatgccta aatctaggc ttgatcagaa gtaatgaac cgttggggcc 16140 ctacattgct atgacatcca aagggccatg atatcagga agaagataa ttacagggt 16200 ctaatgttac agagaggttg agagcagga gatttgatta aaagggtctt tagagctgat 16260 gtcaggtgta tgatgccttt aagagcagtt tttatagtgc agggggtggt caaagagaa 16320 ataggtgct ttctgaggtg acggagcctt gagactagct tatagta actggttt 16380 gtcgtgactt attattctgtg caccaccctg taacatgtac attttattc ctattttcgt 16440 agcatgctct aaagaatggt gatattgtg aaacttcggg taaaccaaaaccacaccta 16500 aaagaaaacc tgctaagaag aaaaagcc aaaagaaa tactgcagct tccttacac 16560 aggttatttt aaatgttga gatttaactt caaggatgt ctcattagtc cttatttaat 16620 agtgtaaaat gtctttaact tagtgatta gtacagtgtt tctattgaca tatactata 16680 caactcaa aacaactatt aaatttctg tattttagga acatgcatat tagtcatgaa 16740 agtataaaga attagatggg aatgaataat gctaaaatca ggacatgtgt tccattgtg 16800 aatggaaggc aggagagg tgccgttgg aaggagtacc aagagccgt aagctgaatt 16860 ggcagtgttt tacatcttaa gctgagagat agattttt ttccctttt tctttaaaaa 16920 ctctaaaact gttaattcca aggaacccag aagtctaggt agattatttc tgctagttaa 16980 aagcagtagt cctgaaagct gatatttg gtgtctttg agccaacttt agttcatca 17040 ttaccaaggg ggaagagagc tacagttga tgagcacttg ctctaggcca gtccagagtg 17100 ctgggcacca tacgcatttt atctccctcc cgctattcac aaaaatatg ggaggtagtt 17160 tatattag ccatctaata agatggggaa actaagactc aaagagattc agaaacttgt 17220 ccatgattat aaatgtaaga gagttggaat tcagatttat gtatttagac cccaagcctt 17280 tctcattaca tcatttgcc tctcattaca tctcattaca tctcattaca tctcattaca tctcattaca caaaacgaga tgagtttg ccctctcaa aagaatgtg tgcatgtata tatctttgat 17400 ttctttgta gtggaaagtt ggggacaaat gttctgccat ttggtcagaa gacggttgca 17460 ttacccagc taccattgct tcattgatt ttagagaga aacctgtgtt gtggtttaca 17520 ctggatatgg aaatagagag gagcaaatc tgtccgatct actttcccca atctgtgaag 17580 tagctataa tatagaaaaatgctcaag agtaaggt aaaaaaa aaaaattcaa 17640 tttctggaag cagagactag atgagaaact gttaaacagt atacacagtt gtcagtttga 17700 tccaccgagg cattaattttt ttcttaatca cacccttata acaaaaacct gcatattttt 17760 tctttttaaa gaatgaaaat gaaagccaag tttcaacaga tgaaagtgag aactccaggt 17820 ctcctggaaa taaatcagat aacatcaagc ccaaatctgc tccatggaac tcttttctcc 17880 ctccaccacc ccccatgcca gggccaagac tgggaccagg aaaggtaaac cttctatgaa 17940 agttttccag aaatagtta atgtcgggac atttaacctc tctgttaact aatttgtagc 18000 tctcccatga aacttttgta gcttaaatac acaagaattt tttgaaaagg aaataagata 18060 atgatgcaaa atagttaatt ttttaaaaaa atgttagaca ctgcagtgga tgcaacaaaa 18120 tactttatat gaaagattta tccagttaac ttttgtggag tattaggtat tagactaata 18180 attagcacac ttacttaagt tagaaagtat aataatgcgc cggacgcggt agctcacgcc 18240 tgtaatccca gcactttggg aggccaaggt gggcggatca caaggtcagg agatcgagac 18300 catcctggct aacacggtga aaccccatct ctactgaaaa tacaaaaaaa tttgccgggc 18360 gtgatggcgg gcacctgtag tcccagctac tcgggaggct gaggcaggag gatggtgtga 18420 accccggagg cagagcttgc agtgagtcaa gatcgtgcca ctgcactcca acctgggcga 18480 cagaatgaga ctccatctca aaaaaaaaa caaaaaaaaaa tgtaataata 18540 attatcatt agctggatga tatgctgttg tttcccatgt cacctgtata agatatgtaa 18600 aataagaaca cattatttac atctaatata gataaaatcc tgaggcgctc tcagattgtt 18660 ttgtagagtt caaatgtaaa tattgttttc attatggtc cttttggtta taagtaacag 18720 aaatcaactc taaaaagatt tttattatag gttagattat gtcatggaac cttaaggctt 18780 gtccctttct agttcttttg tgtaaagcgg tgatttcttc catggaggga atggtattta 18840 ggcaattttt ttttttttt cgagatggag tcttgctctg tcgctcaggc tggagtgcag 18900 tggcaccatt tcagctcact gcaacttcca cctcctgggt tcaagtgatt ctcctgcttc 18960 agcctcccaa gtagctgaga ttacaggcac ccgccaccac acccggctta ttttgtattt 19020 ttagtagaga tggggtttca ccatgttggc caggctggtc ttgaactcct gacctcaagt 19080 gatctcccca ccttggcctt ccaaagtgct aggattacag gcgcctagcc taggcagtca 19140 ttttcaaaaa acaagcatga ctcaccaaaa gttttaagat tttctgtgat aatgttctta 19200 ttgaggctta cattatatta cagtttcttg aatctaaaat gatgtaccct cttagaatat 19260 atacatcatg cttcattggt ctcagggggc tgatttttat aaggagagat ttgctagtttt 19320 tcacaatatg tcctctaagt tggcatgtat agctaaacag gctttcataa aatatacaa 19380 tttagttaat gaaatttggg atatagtctt ttatgattga aataattttg ctaaatagac 19440 tgtctctgat ttattaggta atcaccactc ttattttgtt ttacttcctt aatgtctaca 19500 tagaaaggaa atgagaaaaa tccagaggtt gtcatttgac ttatgagtct gtttgacttc 19560 agatttggt acatgaaatt tcacttaatc tttttgatat gtataaaaca aatattctgg 19620 gtaattattt ttatcctttt ggttttgagt cctttttatt cctatcatat tgaaattggt 19680 aagttaattt tcctttgaaa tattccttat agccaggtct aaattcaat ggccaccac 19740 cgccaccgcc accaccacca cccacttac tatcatgctg gctgcctcca tttccttg 19800 gaccaccagt agtaaaaaa gagtataggt tagattttgc ttcacatac aatttgataa 19860 ttagcagaat agaggattgt aaatgtcat tgtagaacat cccttggggcc agattctaat 19920 gggtagaaat ttgaactaaa cctctgggtt ttgtttgttt ttaatgcctt tctgttaccc 19980 agatgcagtg ctcttgtagt cccaagtcta agctctaggt tgccttctt cctggcagaa 20040 gttggtgtct atgccataag gaggtagttc ctgttagaag ggatttaatt attackttata 20100 taaggaatta gtgtttgccc ttctaggtat agttggatgt tagctctga tgtaaactgg 20160 atttcttttt ctttctctct cttttttttttttgttg gaggcagagt ttgcccttg 20220 taccccaggc tggagtgcag tggtgtgatc tcagctcaca gcaacctccg cctcctgggt 20280 tcaagcaatt ctgcctcggc ctcccaagta gctgggatta caggcgactg ccaccacc 20340 cggctaattt ttgttttatt agtagagatg gggttttcacc atgttggcca gactgatctt 20400 gaactcctga cctcaggtga tccacccgcc ttggcctccc aaagcgctgg gattacaggc 20460 gtgagctgcc gcacccagct gtaaactgga tttctaatgg tagattttta ggtattaaca 20520 atagataaaa agatactttt tggcatactg tgtattggga tggggttaga acaggtgttc 20580 tacccaagac attacttaa aatcgccctc gaaatgctat gtgagctgtg tgtgtgtgtg 20640 tgtgtgtgtg tgtattaagg aaaagcatga aagtatttat gcttgattttt ttttttttac 20700 tcatagcttc atagtggaac agatacatag tctaaatcaa aatgtttaaa ctttttatgt 20760 cacttgctgt cttttcgtcc tcgttaaatt taattttgtt ggtctttttgt tgttattggt 20820 tggttttctc caaatgctag ctatgttaag aaatttaagg ccaggtacag tggctcatgc 20880 ctgtaatccc ggcattttag aaggctgagg caggaggatc acttgagctc aggagtttga 20940 gaccagtctg ggcaacatag caagacctcg tctttgttta ggggaaaaaa aagaaattta 21000 agtaggagat tatataagca aaaatacaat taatttccag cattcactat ataatataaa 21060 tctccagact ttacttttt gtttactgga tataaacaat atctttttct gtctccagat 21120 aattccccca ccacctccca tatgtccaga ttctcttgat gatgctgatg ctttgggaag 21180 tatgttaatt tcatggtaca tgagtggcta tcatactggc tattatatgg taagtaatca 21240 ctcagcatct tttcctgaca atttttttgt agttatgtga ctttgttttg taaatttata 21300 aaatactact tgcttctctc tttatattac taaaaaataa aaataaaaaa atacaactgt 21360 ctgaggctta aattactctt gcattgccc taagtataat tttagttaat tttaaaaagc 21420 tttcatgcta ttgttagatt atttgatta tacacttttg aattgaaatt atactttttc 21480 taaataatgt tttaatctct gatttgaaat tgattgtagg gaatggaaaa gatgggataa 21540 tttttcataa atgaaaaatg aaattcttttt ttttttttttttttga gacggagtct 21600 tgctctgttg cccaggctgg agtgcaatgg cgtgatcttg gctcacagca agctctgcct 21660 cctggattca cgccattctc ctgcctcagc ctcagaggta gctgggacta caggtgcctg 21720 ccaccacgcc tgtctaattt tttgtatttt tttgtaaaga cagggtttca ctgtgttagc 21780 caggatggtc tcaatctcct gaccccgtga tccacccgcc tcggccttcc aagagaaatg 21840 aaattttttt aatgcacaaa gatctggggt aatgtgtacc acattgaacc ttggggagta 21900 tggcttcaaa cttgtcactt tatacgttag tctcctacgg acatgttcta ttgtatttta 21960 gtcagaacat ttaaaattat tttatttat tttatttttt ttttttttt gagacggagt 22020 ctcgctctgt cacccaggct ggagtacagt ggcgcagtct cggctcactg caagctccgc 22080 ctcccgggtt cacgccattc tcctgcctca gcctctccga gtagctggga ctacaggcgc 22140 ccgccaccac gcccggctaa ttttttttta tttttagtag agacggggtt tcaccgtggt 22200 ctcgatctcc tgacctcgtg atccacccgc ctcggcctcc caaagtgctg ggattacaag 22260 cgtgagccac cgcgcccggc ctaaaattat tttaaaagt aagctcttgt gccctgctaa 22320 22380 tgacttaaag gtgtatgttt ttaaatgtat catctgtgtg tgcccccatt aatattctta 22440 tttaaaagtt aaggccagac atggtggctt acaactgtaa tcccaacagt ttgtgaggcc 22560. gaggcaggca gatcacttga ggtcaggagt ttgagaccag cctggccaac atgatgaaac cttgtctcta ctaaaatac caaaaaaat ttagccaggc atggtggcac atgcctgtaa tccgagctac ttgggaggct gtggcagga aattgcttta atctggggagg cagaggttgc agtgagttga gattgtgcca ctgcactcca cccttggtga cagagtgaga ttccatctca aaaaaagaaa aaggcctggc acggtggctc acacctata tcccagtact ttggggaggta gaggcaggtg gatcacttga ggttaggagt tcaggaccag cctggccaac atggtgacta ctccatttct actaataca caaaacttag cccagtggcg ggcagttgta atcccagcta cttgagaggt tgaggcagga gaatcacttg aacctggggag gcagaggttg cagtgagccg agatcacacc gctgcactct agcctggcca acagagtgag aatttgcgga gggaaaaaaa agtcacgctt cagttgttgt agtataacct tggtatattg tatgtatcat gaattcctca ttttaatgac caaaagtaa taaatcaaca gcttgtaatt tgttttgaga tcagttatct gactgtaaca ctgtaggctt ttgtgttttt 23220. gactgtaaca ctgtaggctt ttgtgttttt aatgtatata taaagtattg gtataattta tgttctaat aactttcttg agaaataatt 23340. cacatggtgt gcagtttacc tttgaaagta tacaagttgg ctgggcacaa tggctcacgc ctgtaatccc agcactttgg gaggccaggg caggtggatc acgaggtcag gagatcgaga ccatcctggc taacatggtg aaaccccgtc tctactaaaa gtacaaaac aaattagccg ggcatgttgg cgggcacctt ttgtcccagc tgctcgggag gctgaggcag gagagtggcg 23520 tgaacccagg aggtggagct tgcagtgagc cgagattgtg ccagtgcact ccagcctggg cgacagagcg agactctgtc tcaaaaaata aaataaaaaa gaaagtatac aagtcagtgg ttttggtttt cagttatgca accatcacta caatttaga acattttcat caccccaaaa agaaaccctg ttaccttcat tttccccagc cctaggcagt cagtacactt tctgtctcta 23760. tgaatttgtc tattttagat attatatata aacggaatta tacgatatgt ggtcttttgt gtctggcttc tttcacttag catgctattt tcaagattca tccatgctgt agaatgcacc 23880 agtactgcat tccttcttat tgctgaatat tctgttgttt ggttatatca cattttatcc 23940 attcatcagt tcatggacat ttaggttgtt tttatttttg ggctataatg aataatgttg 24000 ctatgaacat tcgtttgtgt tctttttgtt ttttggtttt tttgggtttt ttttgttttg 24060 tttttgttt tgagacagtc ttgctctgtc tcctaagctg gagtgcagtg gcatgatctt 24120 ggcttactgc aagctctgcc tcccgggttc acaccattct cctgcctcag cccgacaagt 24180 agctgggact acaggcgtgt gccaccatgc acggctaattt ttttgtattt ttagtagaga 24240 tggggttca ccgtgttagc caggatggtc tcgatctcct gacctcgtga tctgcctgcc 24300 taggcctccc aaagtgctgg gattacaggc gtgagccact gcacctggcc ttaagtgttt 24360 ttaatacgtc attgccttaa gctaacaatt cttaaccttt gttctactga agccacgtgg 24420 ttgagatagg ctctgagtct agcttttaac ctctatcttt ttgtcttaga aatctaagca 24480 gaatgcaaat gactaagaat aatgttgttg aaataacata aaataggtta taactttgat 24540 actcattagt aacaatctt tcaatacatc ttacggtctg ttaggtgtag attagtaatg 24600 aagtgggaag ccactgcaag ctagtataca tgttaggaaa gatgaaagc attgaagcca 24660 gagagagac agaggacatt tgggtagat ctgacagaa aaaaaaatgt tttagtatta 24720 atttttgact ttaaattttt ttttatta gtgaatactg gtgtttaatg gtctcatttt 24780 aaagtag acacaggtag tttaggtca tatattttat ttgatgaaaaaggtatag 24840 gccggggcacg gtggctcaca cctgtaatcc cagcacttg gggaggccgag gcaggcggat 24900 cacctgaggt cgggagttag agactagcct siacatggag aaaccccgtc tctactaaaa 24960 aaaatacaaa attaggcgg cgtggtggtg catgcctgta atcccagcta ctcaggaggc 25020 tgaggcagga gattgcttg aacctgggag gtggaggttg cggtgagccg agatcaccctc 25080 attgcactcc agcctgggca acagagcaa aactccatct CAAAAAAAAAAAAAAAAGTT 25140 ataagcggggc tcaggacat cattggacat actgaaagaa gaaaaatcag ctggggcgcag 25200 tggctcacgc cggtaatccc aactttgg gaggccaagg caggcgaatc acctgaagtc 25260 gggagttcca gatcagcctg accaacatgg agaaaccctg tctctactaa aaatacaaaa 25320 ctagccgggc atggtggcgc atgcctgtaa tcccagctac ttgggaggct gaggcaggag 25380 aattgcttga accgagaagg cggaggttgc ggtgagccaa gattgcacca ttgcactcca 25440 gcctgggcaa caagagcgaa actccgtctc aaaaaaaaaa ggaagaaaaa tattttttta 25500 aattaattag tttatttatt ttttaagatg gagttttgcc ctgtcaccca ggctggggtg 25560 caatggtgca atctcggctc actgcaacct ccgcctcctg ggttcaagtg attctcctgc 25620 ctcagcttcc cgagtagctg tgattacagc catatgccac cacgcccagc cagttttgtg 25680 ttttgttttg ttttttgttt tttttttg agagggtgtc ttgctctgtc ccccaagctg 25740 gagtgcagcg gcgcgatctt ggctcactgc aagctctgcc tcccaggttc acaccattct 25800 cttgcctcag cctcccgagt agctgggact acaggtgccc gccaccacac ccggctaatt 25860 ttttgtgtt tttagtagag atggggttc actgtgttag ccaggatggt ctcgatctcc 25920 tgaccttttg atccacccgc ctcagcctcc ccaagtgctg ggattatagg cgtgagccac 25980 tgtgcccggc ctagtcttgt atttttagta gagtcgggat ttctccatgt tggtcaggct 26040 gttctccaaa tccgacctca ggtgatccgc ccgccttggc ctccaaaagt gcaaggcaag 26100 gcattacagg catgagccac tgtgaccggc aatgttttta aattttttac atttaaattt 26160 tattttttag agaccaggtc tcactctatt gctcaggctg gagtgcaagg gcacattcac 26220 agctcactgc agccttgacc tccagggctc aagcagtcct ctcacctcag tttcccgagt 26280 agctgggact acagtgataa tgccactgca cctggctaat ttttattttt atttatttat 26340 ttttttttga gacagagtct tgctctgtca cccaggctgg agtgcagtgg tgtaaatctc 26400 agctcactgc agcctccgcc tcctgggttc aagtgattct cctgcctcaa cctcccaagt 26460 agctgggatt agaggtcccc accaccatgc ctggctaatt ttttgtactt tcagtagaaa 26520 cggggttttg ccatgttggc caggctgttc tcgaactcct gagctcaggt gatccaactg 26580 tctcggcctc ccaaagtgct gggattacag gcgtgagcca ctgtgcctag cctgagccac 26640 cacgccggcc taatttttaa atttttgta gagacagggt ctcattatgt tgcccagggt 26700 ggtgtcaagc tccaggtctc aagtgatccc cctacctcg cctcccaaag ttgtgggatt 26760 gtaggcatga gccactgcaa gaaaacctta actgcagcct aataattgtt ttctttggga 26820 taactttaa agtacattaa aagactatca acttaatttc tgatcatatt ttgttgaata 26880 aaataagtaa aatgtctttgt gaaacaaaat gctttttaac atccatataa agctatctat 26940 atatagctat ctatatctat atagctattt ttttaactt cctttatttt ccttacaggg 27000 ttttagacaa aatcaaaaag aaggaaggtg ctcacattcc ttaaattaag gagtaagtct 27060 gccagcatta tgaaagtgaa tcttactttt gtaaaacttt atggtttgtg gaaaacaaat 27120 gttttgaac atttaaaaag ttcagatgtt agaaagttga aaggttaatg taaaacaatc 27180 aatattaaag aatttgatg ccaaaactat tagataaaag gttaatctac atccctacta 27240 gaattctcat acttaactgg ttggttgtgt ggaagaaaca tactttcaca ataaagagct 27300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttaggatatg atgccatttt atatcactag taggcagacc agcagacttt tttttattgt 27360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatatgggat aacctaggca tactgcactg tacactctga catatgaagt gctctagtca 27420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agtttaactg gtgtccacag aggacatggt ttaactggaa ttcgtcaagc ctctggttct 27480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aatttctcat ttgcaggaaa tgctggcata gagcagcact aaatgacacc actaaagaaa 27540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgatcagaca gatctggaat gtgaagcgtt atagaagata actggcctca tttcttcaaa 27600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atatcaagtg ttgggaaaga aaaaaggaag tggaatggggt aactcttctt gattaaaagt 27660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tatgtaataa ccaaatgcaa tgtgaaatat tttactggac tctattttga aaaaccatct 27720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtaaaagact gaggtggggg tgggaggcca gcacggtggt gaggcagttg agaaaatttg 27780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aatgtggatt agattttgaa tgatattgga taattattgg taattttatg agctgtgaga 27840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agggtgttgt agtttataaa agactgtctt aatttgcata cttaagcatt taggaatgaa 27900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtgttagagt gtcttaaaat gtttcaaatg gtttaacaaa atgtatgtga ggcgtatgtg 27960<h2 style=";text-align:left;direction:ltr"> gcaaaatgtt acagaatcta actggtggac atggctgttc attgtactgt ttttttctat 28020 cttctatatg tttaaaagta tataataaaa atatttaatt tttttttaaa 28070 <210> 2 <400> 2 000 <210> 3 <400> 3 000 <210> 4 <400> 4 000 <210> 5 <400> 5 000 <210> 6 <400> 6 000 <210> 7 <400> 7 000 <210> 8 <400> 8 000 <210> 9 <400> 9 000 <210> 10 <400> 10 000 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 11 gctgatgctt tgggaagtat gtta 24 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 12 caccttcctt ctttttgatt ttgtc 25 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Probe <400> 13 tacatgagtgctatcatac t 21 <210> 14 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 14 catggtacat gagtggctat catactg 27 <210> 15 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 15 tggtgtcatt tagtgctgct ctatg 25 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Probe <400> 16 ccagcatttc catataatag c 21 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 caggaggatt ccgtgctgtt 20 <210> 18 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 cagtgctgta tcatcccaaa tgtc 24 <210> 19 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 19 acaggccaga gcgat 15 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 20 attcactttc ataatgctgg 20 <210> twenty one <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty one cactttcata atgctggc 18 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty two ttcactttca taatgctggc 20 <210> twenty three <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty three tcactttcat aatgctgg 18 <210> twenty four <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty four actttcataa tgctggcag 19 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 25 cactttcata atgctggcag 20 <210> 26 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 26 cactttcata atgctggca 19 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 27 tcactttcat aatgctggca 20 <210> 28 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 28 ctttcataat gctggc 16 <210> 29 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 29 actttcataa tgctggc 17 <210> 30 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 30 tcactttcat aatgctggc 19 <210> 31 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 31 actttcataa tgctgg 16 <210> 32 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 32 cactttcata atgctgg 17 <210> 33 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 33 ttcactttca taatgctgg 19 <210> 34 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 34 attcactttc ataatgctg 19 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 35 agattcactt tcataatgct 20 <210> 36 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 36 tcactttcat aatgctggt 19 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 37 tcactttcat aatgctgga 19 <210> 38 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> misc_feature <222> (19)..(19) <223> The bases at these positions are RNA <400> 38 tcactttcat aatgctggu 19 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 39 tcactttcat aatgctggaa 20 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 40 tcactttcat aatgctggat 20 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 41 tcactttcat aatgctggac 20 <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 42 tcactttcat aatgctggtc 20 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 43 tcactttcat aatgctggtt 20 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 44 tcactttcat aatgctggta 20 <210> 45 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 45 tcactttcat aatgctggcc 20 <210> 46 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 46 tcactttcat aatgctggct 20 <210> 47 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 47 ctcactttca taatgctgg 19 <210> 48 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 48 atcactttca taatgctgg 19 <210> 49 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 49 gattcacttt cataatgctg 20 <210> 50 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 50 gattcacttt cataatgct 19

Claims

1. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO: 21), or a salt thereof.

2. The modified oligonucleotide of claim 1, wherein the salt of the modified oligonucleotide comprises a sodium salt or a potassium salt.

3. A modified oligonucleotide according to the following chemical structure: (SEQ ID NO:21).

4. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: m C ns A no m C ns T no T ns T ns m C ns A ns T ns A ns A ns T ns G ns m C ns T ns G ns G ns m C n (SEQ ID NO:21), wherein: A = adenine nucleobase, m C=5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, n=2'-NMA sugar moiety, s = phosphorothioate internucleoside linkage, and o = phosphodiester internucleoside linkage.

5. A population of modified oligonucleotides as described in any one of claims 1 to 3, wherein all phosphorothioate internucleoside linkages of the modified oligonucleotides are stereo-random.

6. A population of oligomeric compounds as claimed in claim 4, wherein all phosphorothioate internucleoside linkages of the modified oligonucleotides are stereo-random.

7. A pharmaceutical composition comprising the modified oligonucleotide of any one of claims 1 to 3, the oligomeric compound of claim 4, a population of modified oligonucleotides of claim 5 or a population of oligomeric compounds of claim 6, and a pharmaceutically acceptable diluent.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable diluent is aCSF or PBS.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises the modified oligonucleotide of any one of claims 1 to 3 and aCSF.

10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises the modified oligonucleotide of any one of claims 1 to 3 and PBS.

11. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises the oligomeric compound of claim 4 and aCSF.

12. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises the oligomeric compound of claim 4 and PBS.

13. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises a population of modified oligonucleotides of claim 5 and aCSF.

14. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises a population of modified oligonucleotides as claimed in claim 5 and PBS.

15. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises a population of oligomeric compounds of claim 6 and aCSF.

16. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises a population of oligomeric compounds of claim 6 and PBS.

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