JPWO2020023737A5 - - Google Patents

Description

SEQUENCE LISTING This application is being filed in electronic form along with a sequence listing. The Sequence Listing is BIOL0321WOSEQ_ST25. txt file name. The information in electronic form of the Sequence Listing is hereby incorporated by reference in its entirety.

Compounds, methods, and pharmaceutical compositions are provided for reducing the amount or activity of ATXN2 RNA in a cell or animal, and in certain examples, for reducing the amount of ataxin-2 protein in a cell or animal. . Such compounds, methods, and pharmaceutical compositions are useful for ameliorating at least one symptom or feature of neurodegenerative diseases. Such symptoms and features include ataxia, neuropathy, and aggregate formation. Such neurodegenerative diseases include spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and parkinsonism.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disorder characterized by progressive neuronal function and cell loss in the cerebellum, brainstem, and spinal cord. The cause of SCA2 is a CAG expansion in the ATXN2 gene that results in a polyglutamine (polyQ) expansion in the ataxin-2 protein. Patients with SCA2 are characterized by progressive cerebellar ataxia, slow saccadic eye movements, and other neurological features such as neuropathy (Pulst, SM (ed.), Genetics of Movement Disorders. Elsevier, Inc., Amsterdam, 2003, pp. 19-34.). A moderate CAG expansion in the ATXN2 gene is also associated with parkinsonism or amyotrophic lateral sclerosis (ALS), which is indistinguishable from idiopathic forms of these diseases (Kim et al., Arch. Neurol., 2007, 64: 1510-1518, Ross et al., Hum. Mol. Genet., 2011, 20: 3207-3212, Corrado et al., Hum. , Nature, 2010, 466:1069-1075; Van Damme et al., Neurology, 2011, 76:2066-2072).

The pathogenic function of polyQ disease proteins that accompanies polyQ expansion may result from the development of nuclear inclusions or the acquisition of toxicity associated with soluble toxic oligomers (Lajoie et al., PLoS One, 2011, 5:e15245). The brains of SCA2 patients are characterized by loss of Purkinje cells, whereas SCA2 Purkinje cells are deficient in inclusion bodies, suggesting that polyQ-extended ataxin-2, which is not associated with inclusion formation, can cause toxicity. (Huynh et al., Ann. Neurol., 1999, 45:232-241). The functions obtained with polyQ-elongated ataxin-2 include abnormal accumulation of the Golgi (Huynh et al., Hum. Mol. Genet., 2003, 12:1485-1496), normal gain of function (Duvick et al., Neuron , 2010, 67:929-935), and sequestration of transcription factors (TFs) such as other polyQ proteins and glyceraldehyde-3-phosphate dehydrogenase (Yamanaka et al., Methods Mol. Biol., 2010:648, 215). -229, Koshy et al., Hum. Mol. Genet., 1996, 5:1311-1318, Burke et al., Nat. Med., 1996, 2:347-350). Several normal functions of ataxin-2 have been characterized. Ataxin-2 is present in stress granules and P-bodies, suggesting a function in sequestering mRNA and in regulating protein translation during stress (Nonhoff et al., Mol. Biol. Cell, 2007, 18:1385- 1396). Overexpression of ataxin-2 interfered with P-body assembly, whereas underexpression interfered with stress granule assembly (Nonhoff et al., Mol. Biol. Cell, 2007, 18:1385-1396). Interactions with polyA-binding protein 1, the RNA splicing factor A2BP1/Fox1, and polyribosomes further support a role for ataxin-2 in RNA metabolism (Shibata et al., Hum. Mol. Genet., 2000, 9 : 1303-1313, Ciosk et al., Development, 2004, 131:4831-4841, Satterfield et al., Hum. Mol. Ataxin-2 is a regulator of EGF receptor internalization and signaling through its interaction with SRC kinase and the endocytic protein CIN85. (Nonis et al., Cell Signal., 2008, 20:1725-1739). Ataxin-2 also interacts with the ALS-associated protein TDP-43 in an RNA-dependent manner, and familial and sporadic ALS are associated with the development of long normal CAG repeat expansions ATXN2 (Elden et al., Nature, 2010). , 466:1069-1075; Van Damme et al., Neurology, 2011, 76:2066-2072).

There is currently a lack of acceptable options for treating such neurodegenerative diseases. It is therefore the goal herein to provide methods for the treatment of such diseases.

To reduce the amount or activity of ATXN2 RNA, in certain embodiments, provided herein are compounds, methods, and pharmaceutical compositions for reducing the amount of ataxin-2 protein in a cell or animal. . In certain embodiments, the animal has a neurodegenerative disease. In certain embodiments, the animal has Spinocerebellar Ataxia Type 2 (SCA2), Amyotrophic Lateral Sclerosis (ALS), and Parkinsonism. In certain embodiments, compounds useful for reducing ATXN2 RNA expression are oligomeric compounds. In certain embodiments, oligomeric compounds comprise modified oligonucleotides.

Also provided are methods useful for ameliorating at least one symptom or characteristic of a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is SCA2, ALS, or Parkinsonism. In certain embodiments, symptoms and features include ataxia, neuropathy, and aggregate formation. In certain embodiments, amelioration of these symptoms results in improved motor function, reduced neuropathy, and reduced numbers of aggregates.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. In this specification, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising two or more subunits, unless otherwise specified. do.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, recited in this application, including, but not limited to, patents, patent applications, articles, books, and articles, are part of the documents discussed herein, and their entireties are expressly incorporated herein by reference.

DEFINITIONS Unless specific definitions are provided, the terminology used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, and procedures and techniques thereof, described herein are those of skill in the art. well known in the art and commonly used. Where permitted, all patents, applications, published applications, and other publications and other data referenced throughout this disclosure are herein incorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the following meanings.
As used herein, "2'-deoxynucleoside" means a nucleoside containing a 2'-H(H) deoxyribosyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). do. In certain embodiments, 2'-deoxynucleosides may include modified nucleobases or may include RNA nucleobases (uracil).

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

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

As used herein, "administering" means providing a pharmaceutical agent to an animal.

As used herein, "animal" means either human or non-human animals.

As used herein, "antisense activity" means any detectable and/or measurable change that can result from the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a reduction in the amount or expression of a target nucleic acid or a protein encoded by such a target nucleic acid compared to the target nucleic acid or target protein levels in the absence of the antisense compound. be.

As used herein, "antisense compound" means an oligomeric compound capable of achieving at least one antisense activity.

As used herein, "ameliorate" in relation to treatment means improvement in at least one symptom for the same symptom in the absence of treatment. In certain embodiments, the amelioration is a reduction in the severity or frequency of symptoms, or a delay in the onset of symptoms, or a delay in the progression of severity or frequency of symptoms. In certain embodiments, the symptoms or features are ataxia, neuropathy, and aggregate formation. In certain embodiments, amelioration of these symptoms results in improved motor function, reduced neuropathy, or reduced number of aggregates.

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

As used herein, "bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety that contains two rings, the second ring is formed through a bridge that connects two of the atoms in, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, bicyclic sugar moieties do not include furanosyl moieties.

As used herein, "cleavable moiety" means a bond or group of atoms that is cleaved under physiological conditions, eg, in a cell, animal, or human.

As used herein, "complementary" in relation to an oligonucleotide means at least 70 nucleobases of an oligonucleotide or one or more regions thereof and another nucleic acid or one or more regions thereof. The % means that the nucleobase sequences of the oligonucleotide and the other nucleic acid can hydrogen bond with each other when aligned in opposite directions. Complementary nucleobases refer to nucleobases that are capable of forming hydrogen bonds with each other. Complementary nucleobase pairs are adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methylcytosine (mC) and guanine (G). include. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some incompatibilities are tolerated. As used herein, "fully complementary" or "100% complementary" in relation to an oligonucleotide means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide. It means that there is

As used herein, " conjugate group " means a group of atoms that are directly attached to an oligonucleotide. A conjugate group includes a conjugate moiety and a conjugate linker that joins the conjugate moiety to the oligonucleotide.

As used herein, " conjugate linker" means a single bond or group of atoms comprising at least one bond connecting a conjugate moiety to an oligonucleotide.

As used herein, " conjugate moiety" means a group of atoms attached to an oligonucleotide via a conjugate linker.

As used herein, "adjacent" in the context of oligonucleotides refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to each other in a sequence.

As used herein, "constrained ethyl" or "cEt" or "cEt-modified sugar" means a β-D ribosyl bicyclic sugar moiety, in which the second ring of the bicyclic sugar is formed through a bridge connecting the 4′-carbon and the 2′-carbon of the β-D ribosyl sugar moiety, the bridge having the formula 4′—CH(CH ₃ )—O-2′ and the bridge is in the S configuration.

As used herein, "cEt nucleoside" means a nucleoside containing a cEt-modified sugar.

As used herein, a "chirally enriched population" means a plurality of molecules of identical molecular formula, wherein the number of molecules within the population that contain a particular stereochemical configuration at a particular chiral center. The number or percentage is greater than the number or percentage of molecules within a population that would be expected to contain the same particular stereochemical configuration at the same particular chiral center if the particular chiral center were stereorandom. A chirally enriched population of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound comprising modified oligonucleotides.

As used herein, a "gapmer" is a modified oligonucleotide comprising an internal region with multiple nucleosides that supports RNase H cleavage located between an external region with one or more nucleosides. and a nucleoside containing an internal region is chemically distinct from a nucleoside or a nucleoside containing an external region. The inner region may be referred to as the "gap" and the outer region may be referred to as the "wing". Unless otherwise indicated, "gapmers" refer to sugar motifs. Unless otherwise indicated, the sugar moieties of the gap nucleosides of gapmers are unmodified 2'-deoxyribosyl. Thus, "MOE gapmer" refers to a gapmer with 2'-MOE nucleoside sugar motifs in both the wings and the 2'-deoxynucleoside gap. Unless otherwise indicated, MOE gapmers may include one or more modified internucleoside linkages and/or modified nucleobases, and such modifications do not necessarily follow the gapmer's pattern of sugar modifications. is not.

As used herein, a "hotspot region" is a range of nucleobases of a target nucleic acid that is compatible with oligomeric compound-mediated reduction of the amount or activity of the target nucleic acid.

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

As used herein, the term "internucleoside linkage" is a covalent bond between adjacent nucleosides within an oligonucleotide. As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage. A "phosphorothioate internucleoside linkage" is a modified internucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester internucleoside linkage is replaced with a sulfur atom.

As used herein, "linker nucleoside" means a nucleoside that either directly or indirectly links an oligonucleotide to a conjugate moiety. A linker nucleoside is placed within the conjugate linker of the oligomeric compound. Linker nucleosides are not considered part of the oligonucleotide portion of the oligomeric compound, even if they are adjacent to the oligonucleotide.

As used herein, a "non-bicyclic modified sugar moiety" includes modifications such as substitutions that do not form a bridge between the two atoms of the sugar to form a second ring. It refers to a modified sugar moiety.

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

As used herein, "MOE" means methoxyethyl. "2'-MOE" or "2'-MOE modified sugar" means a 2'-OCH ₂ CH ₂ OCH ₃ group in place of the 2'-OH group of a ribosyl sugar moiety. As used herein, "2'-MOE nucleoside" means a nucleoside containing a 2'-MOE modified sugar.

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

As used herein, "neurodegenerative disease" means a condition characterized by progressive loss of function or structure, including loss of motor function and death of nerve cells. In certain embodiments, the neurodegenerative disease is spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and parkinsonism.

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 an atom other than unmodified A, T, C, U, or G that can pair with at least one unmodified nucleobase. group. A "5-methylcytosine" is a modified nucleobase. A common base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, "nucleobase sequence" means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modifications.

As used herein, "nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moieties are each independently unmodified or modified. As used herein, "modified nucleoside" means a nucleoside that contains modified nucleobases and/or modified sugar moieties. Modified nucleosides include abasic nucleosides that lack a nucleobase. "Bound nucleosides" are nucleosides that are joined in a contiguous sequence (ie, no additional nucleosides are present between those to which they are joined).

As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more additional features such as a conjugate group or terminal group. The oligomeric compounds may or may not be paired with a second oligomeric compound that is complementary to the first oligomeric compound. A "single-stranded oligomeric compound" is an unpaired oligomeric compound. The term "oligomeric duplex" 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 "double-stranded oligomeric compound."

As used herein, "oligonucleotide" means a chain of linked nucleosides connected via internucleoside linkages, each nucleoside and internucleoside linkage being modified or modified It has not been. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide in which at least one nucleoside or internucleoside linkage has been modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not contain any nucleoside or internucleoside modifications.

As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to animals. Certain such carriers can be used in pharmaceutical compositions such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. Allow to be formulated. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffered solutions, or sterile artificial cerebrospinal fluid.

As used herein, "pharmaceutically acceptable salt" means physiologically and pharmaceutically acceptable salts of a compound. A pharmaceutically acceptable salt retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereon.

As used herein, "pharmaceutical composition" means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition can comprise an oligomeric compound and a sterile aqueous solution. In certain embodiments, the pharmaceutical composition exhibits activity in a free uptake assay in certain cell lines.

As used herein, "prodrug" means a therapeutic agent that is in an extracorporeal form that is converted to a different form within an animal or its cells. Typically, conversion of prodrugs in animals is facilitated by the action of enzymes (eg, endogenous or viral enzymes) or chemicals present in cells or tissues and/or by physiological conditions.

As used herein, "reducing or inhibiting the amount or activity" refers to reducing or blocking transcriptional expression or activity relative to transcriptional expression or activity in an untreated or control sample, not necessarily transcriptional expression or activity. does not indicate the total exclusion of

As used herein, "RNAi compound" means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or the protein encoded by the target nucleic acid. do. RNAi compounds include, but are not limited to, double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimetics. In certain embodiments, RNAi compounds modulate the amount, activity, and/or splicing of a target nucleic acid. The term RNAi compound excludes antisense compounds that act through RNase H.

As used herein, "self-complementary" in relation to an oligonucleotide means an oligonucleotide that hybridizes at least partially to itself.

As used herein, "standard cell assay" means the assay described in Example 3 and reasonable variations thereof.

As used herein, "standard in vivo assay" means the experiment described in Example 15, and reasonable variations thereof.

As used herein, "stereorandom chiral center" in the context of a population of molecules of identical molecular formula means a chiral center having random stereochemical configurations. For example, in a population of molecules containing stereorandom chiral centers, the number of molecules with the (S) configuration of the stereorandom chiral centers can be the same as the number of molecules with the (R) configuration of the stereorandom chiral centers, not necessarily the same. The stereochemical configuration of a chiral center is considered random when it results from synthetic methods not designed to control stereochemical configuration. In certain embodiments, stereorandom chiral centers are stereorandom phosphorothioate internucleoside linkages.

As used herein, "sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" refers to a 2'-OH(H) ribosyl moiety as found in RNA ("unmodified RNA sugar moiety"), or DNA ("unmodified DNA sugar moiety") means a 2'-H(H) deoxyribosyl moiety as found in . The 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.

As used herein, a "sugar surrogate" is a modified nucleobase other than a furanosyl moiety that can attach a nucleobase to another group, such as an internucleoside linkage, a conjugate group , or a terminal group in an oligonucleotide. means the sugar moiety. Modified nucleosides containing sugar surrogates can be conjugated at one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary oligomeric compounds or target nucleic acids.

As used herein, "target nucleic acid" and "target RNA" mean a nucleic acid that an antisense compound is designed to affect.

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

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

As used herein, "therapeutically effective amount" means that amount of pharmaceutical agent that provides a therapeutic effect in an animal. For example, a therapeutically effective amount ameliorates symptoms of a disease.

Certain Specific Embodiments The present disclosure provides the following non-limiting numbered embodiments.
Embodiment 1. An oligomeric compound comprising a modified oligonucleotide consisting of 12-50 linked nucleosides, wherein the nucleobase sequence of said modified oligonucleotide is at least 90% an isometric portion of an ATXN2 nucleic acid. Said oligomeric compound which is complementary and wherein said modified oligonucleotides comprise at least one modification selected from modified sugars, sugar surrogates and modified internucleoside linkages.

Embodiment 2. At least 12, at least 13, at least 14, at least 15, at least 16 of any of the nucleobase sequences of SEQ ID NOS: 30-3319 consisting of 12-50 linked nucleosides , a modified oligonucleotide having a nucleobase sequence comprising at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases.

Embodiment 3. Consisting of 12-50 linked nucleosides, 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 1. An oligomeric compound comprising a modified oligonucleotide having a nucleobase sequence comprising a portion of at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases, said portion comprising:
an isometric portion of nucleobases 2,455 to 2,483 of SEQ ID NO: 1;
an isometric portion of nucleobases 4,393 to 4,424 of SEQ ID NO: 1;
an isometric portion of nucleobases 4,413 to 4,437 of SEQ ID NO: 1;
an isometric portion of nucleobases 4,525 to 4,554 of SEQ ID NO:2;
an isometric portion of nucleobases 4,748 to 4,771 of SEQ ID NO:2;
an isometric portion of nucleobases 9,927 to 9,954 of SEQ ID NO:2;
an isometric portion of nucleobases 10,345 to 10,368 of SEQ ID NO:2;
an isometric portion of nucleobases 17,153 to 17,182 of SEQ ID NO:2;
an isometric portion of nucleobases 18,680 to 18,702 of SEQ ID NO:2;
an isometric portion of nucleobases 23,251 to 23,276 of SEQ ID NO:2;
an isometric portion of nucleobases 28,081 to 28,105 of SEQ ID NO:2;
an isometric portion of nucleobases 28,491 to 28,526 of SEQ ID NO:2;
an isometric portion of nucleobases 28,885 to 28,912 of SEQ ID NO:2;
an isometric portion of nucleobases 32,328 to 32,352 of SEQ ID NO:2;
an isometric portion of nucleobases 32,796 to 32,824 of SEQ ID NO:2;
an isometric portion of nucleobases 32,809 to 32,838 of SEQ ID NO:2;
an isometric portion of nucleobases 36,308 to 36,334 of SEQ ID NO:2;
an isometric portion of nucleobases 36,845 to 36,872 of SEQ ID NO:2;
an isometric portion of nucleobases 49,147 to 49,173 of SEQ ID NO:2;
an isometric portion of nucleobases 57,469 to 57,494 of SEQ ID NO:2;
an isometric portion of nucleobases 82,848 to 82,874 of SEQ ID NO:2;
an isometric portion of nucleobases 83,784 to 83,813 of SEQ ID NO:2;
an isometric portion of nucleobases 84,743 to 84,782 of SEQ ID NO:2;
an isometric portion of nucleobases 84,813 to 84,839 of SEQ ID NO:2;
an isometric portion of nucleobases 85,051 to 85,076 of SEQ ID NO:2;
an isometric portion of nucleobases 97,618 to 97,643 of SEQ ID NO:2;
an isometric portion of nucleobases 119,023 to 119,048 of SEQ ID NO:2;
an isometric portion of nucleobases 132,161 to 132,195 of SEQ ID NO:2;
Said oligomeric compound which is complementary to an isometric portion of nucleobases 139,271 to 139,303 of SEQ ID NO:2 or to an isometric portion of nucleobases 1,075 to 1,146 of SEQ ID NO:1.

Embodiment 4. Said modified oligonucleotide is at least 80%, at least 85%, at least 90% with either the nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2 when measured over the entire nucleobase sequence of said modified oligonucleotide The oligomeric compound of any one of embodiments 1-3, having nucleobase sequences that are 100%, at least 95%, or 100% complementary.

Embodiment 5. The oligomeric compound of any of embodiments 1-4, wherein said modified oligonucleotide comprises at least one modified nucleoside.

Embodiment 6. 6. The oligomeric compound of embodiment 5, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a modified sugar moiety.

Embodiment 7. 7. The oligomeric compound of embodiment 6, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety.

Embodiment 8. Said modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety with a 2'-4' bridge, said 2'-4' bridge being -O-CH2- and -O An oligomeric compound according to embodiment 7, selected from -CH(CH3)-.

Embodiment 9. An oligomeric compound according to any of embodiments 5-8, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety.

Embodiment 10. wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety comprising a 2'-MOE modified sugar or a 2'-OMe modified sugar. An oligomeric compound according to Form 9.

Embodiment 11. An oligomeric compound according to any of embodiments 5-10, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a sugar surrogate.

Embodiment 12. 12. The oligomeric compound of embodiment 11, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising sugar surrogates selected from morpholinos and PNAs.

Embodiment 13. The modified oligonucleotide is
a 5' region consisting of 1-5 linked 5' region nucleosides;
having a sugar motif comprising a central region consisting of 6-10 linked central region nucleosides and a 3' region consisting of 1-5 linked 3' region nucleosides;
Embodiment 1, wherein each of said 5' region nucleosides and each of said 3' region nucleosides comprises a modified sugar moiety and each of said central region nucleosides comprises an unmodified 2'-deoxyribosyl sugar moiety. 13. The oligomeric compound according to any one of -12.

Embodiment 14. 14. The oligomeric compound of any of embodiments 1-13, wherein said modified oligonucleotide comprises at least one modified internucleoside linkage.

Embodiment 15. 15. The oligomeric compound of embodiment 14, wherein each internucleoside linkage of said modified oligonucleotide is a modified internucleoside linkage.

Embodiment 16. 16. An oligomeric compound according to embodiment 14 or 15, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage.

Embodiment 17. 17. The oligomeric compound of embodiment 14 or 16, wherein said modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.

Embodiment 18. 18. The oligomeric compound of any of embodiments 14, 16, or 17, wherein each internucleoside linkage is either a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.

Embodiment 19. 19. The oligomeric compound of any of embodiments 1-18, wherein said modified oligonucleotide comprises at least one modified nucleobase.

Embodiment 20. The oligomeric compound of embodiment 19, wherein said modified nucleobase is 5-methylcytosine.

Embodiment 21. A practice wherein said modified oligonucleotide consists of 12-30, 12-22, 12-20, 14-20, 15-25, 16-20, 18-22, or 18-20 linked nucleosides. An oligomeric compound according to any of Forms 1-20.

Embodiment 22. The oligomeric compound of any of embodiments 1-21, wherein said modified oligonucleotide consists of 18 or 20 linked nucleosides.

Embodiment 23. 23. The oligomeric compound of any of embodiments 1-22, consisting of said modified oligonucleotide.

Embodiment 24. An oligomeric compound according to any of embodiments 1-22, comprising a conjugate group comprising a conjugate moiety and a conjugate linker.

Embodiment 25. 25. The oligomeric compound of embodiment 24, wherein said conjugate group comprises a GalNAc cluster comprising 1-3 GalNAc ligands.

Embodiment 26. 26. The oligomeric compound of embodiment 24 or 25, wherein said conjugate linker consists of a single bond.

Embodiment 27. The oligomeric compound of embodiment 25, wherein said conjugate linker is cleavable.

Embodiment 28. 28. The oligomeric compound of embodiment 27, wherein said conjugate linker comprises 1-3 linker nucleosides.

Embodiment 29. The oligomeric compound of any of embodiments 24-28, wherein said conjugate group is attached to said modified oligonucleotide at the 5' end of said modified oligonucleotide.

Embodiment 30. The oligomeric compound of any of embodiments 24-28, wherein said conjugate group is attached to said modified oligonucleotide at the 3' end of said modified oligonucleotide.

Embodiment 31. An oligomeric compound according to any of embodiments 1-30, comprising terminal groups.

Embodiment 32. The oligomeric compound of any of embodiments 1-31, wherein said oligomeric compound is a single stranded oligomeric compound.

Embodiment 33. The oligomeric compound of any of embodiments 1-27 or 29-31, wherein said oligomeric compound does not comprise a linker nucleoside.

Embodiment 34. An oligomeric duplex comprising an oligomeric compound according to any of embodiments 1-31 or 33.

Embodiment 35. An antisense compound comprising or consisting of an oligomeric compound according to any of embodiments 1-33 or an oligomeric duplex according to embodiment 34.

Embodiment 36. A pharmaceutical composition comprising an oligomeric compound according to any of embodiments 1-33 or an oligomeric duplex according to embodiment 34 and a pharmaceutically acceptable carrier or diluent.

またはその塩。 Embodiment 37. A modified oligonucleotide (SEQ ID NO: 1714) of the formula

Or its salt.

またはその塩。 Embodiment 38. A modified oligonucleotide (SEQ ID NO: 1255) of the formula

Or its salt.

またはその塩。 Embodiment 39. A modified oligonucleotide (SEQ ID NO: 1185) of the formula

Or its salt.

またはその塩。 Embodiment 40. Modified oligonucleotide (SEQ ID NO: 3235) of the formula

Or its salt.

またはその塩。 Embodiment 41. Modified oligonucleotide (SEQ ID NO: 158) of the formula

Or its salt.

またはその塩。 Embodiment 42. Modified oligonucleotide (SEQ ID NO: 2544) of the formula

Or its salt.

Embodiment 43. 43. The modified oligonucleotide of any of embodiments 37-42, which is the sodium salt of said formula.

Embodiment 44. A modified oligonucleotide (SEQ ID NO: 1714) of the formula:

Embodiment 45. A modified oligonucleotide (SEQ ID NO: 1255) of the formula:

Embodiment 46. A modified oligonucleotide (SEQ ID NO: 1185) of the formula:

Embodiment 47. A modified oligonucleotide (SEQ ID NO: 3235) of the formula:

Embodiment 48. A modified oligonucleotide (SEQ ID NO: 158) of the formula:

Embodiment 49. A modified oligonucleotide (SEQ ID NO: 2544) of the formula:

Embodiment 50. 50. A chirally enriched population of modified oligonucleotides according to any of embodiments 37-49, wherein said population has at least one specific phosphorothioate internucleoside linkage with a specific stereochemical configuration. Said chirally enriched population enriched for modified oligonucleotides comprising:

Embodiment 51. 51. The chirally enriched population of embodiment 50, wherein said population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) configuration.

Embodiment 52. 52. A chirally enriched population according to embodiment 50 or 51, wherein said population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Rp) configuration.

Embodiment 53. 51. A chirally enriched population according to embodiment 50, wherein said population is enriched for modified oligonucleotides having specific, independently selected stereochemical configurations at each phosphorothioate internucleoside linkage.

Embodiment 54. 54. The chirally enriched population of embodiment 53, wherein said population is enriched for modified oligonucleotides having said (Sp) configuration at each phosphorothioate internucleoside linkage.

Embodiment 55. 54. The chirally enriched population of embodiment 53, wherein said population is enriched for modified oligonucleotides having said (Rp) configuration at each phosphorothioate internucleoside linkage.

Embodiment 56. Embodiment 50 or Embodiment 53, wherein said population is enriched for modified oligonucleotides having at least 3 adjacent phosphorothioate internucleoside linkages in the Sp-Sp-Rp configuration in the 5' to 3' direction. A chirally enriched population as described in .

Embodiment 57. 50. The population of modified oligonucleotides according to any of embodiments 37-49, wherein all of said phosphorothioate internucleoside linkages of said modified oligonucleotides are stereorandom. group.

Embodiment 58. A pharmaceutical composition comprising the modified oligonucleotide of any of embodiments 37-49 and a pharmaceutically acceptable diluent or carrier.

Embodiment 59. 59. The pharmaceutical composition of embodiment 58, wherein said pharmaceutically acceptable diluent is artificial cerebrospinal fluid.

Embodiment 60. 60. The pharmaceutical composition of embodiment 59, wherein said pharmaceutical composition consists essentially of said modified oligonucleotide and artificial cerebrospinal fluid.

Embodiment 61. A method comprising administering the pharmaceutical composition of any of embodiments 36 or 58-60 to an animal.

Embodiment 62. A method of treating a disease associated with ATXN2, comprising administering to an individual having or at risk of developing a disease associated with ATXN2 a therapeutically effective amount of any of embodiments 36 or 58-60. and thereby treating said disease associated with ATXN2.

Embodiment 63. 63. The method of embodiment 62, wherein said disease associated with ATXN2 is a neurodegenerative disease.

Embodiment 64. 64. The method of embodiment 63, wherein the neurodegenerative disease is any of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and parkinsonism.

Embodiment 65. 65. The method of embodiment 64, wherein at least one symptom or feature of said neurodegenerative disease is ameliorated.

Embodiment 66. 66. The method of embodiment 65, wherein the symptom or feature is any of ataxia, neuropathy, and aggregate formation.

Embodiment 67. An oligomeric compound comprising a modified oligonucleotide of the formula:
Ges Teo Aeo mCeo Teo Tds Tds Tds mCds Tds mCds Ads Tds Gds Tds Geo mCeo Ges Ges mCe (SEQ ID NO: 1714), wherein
A = adenine nucleobase,
mC = 5 -methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
Said oligomeric compound, wherein s = phosphorothioate internucleoside linkages and o = phosphodiester internucleoside linkages.

Embodiment 68. An oligomeric compound comprising a modified oligonucleotide of the formula mCes Teo Geo mCeo Tds Ads Ads mCds Tds Gds Gds Tds Tds Tds Geo mCeo mCeo mCes
Tes Te (SEQ ID NO: 1255), where
A = adenine nucleobase,
mC = 5 -methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
Said oligomeric compound, wherein s = phosphorothioate internucleoside linkages and o = phosphodiester internucleoside linkages.

Embodiment 69. An oligomeric compound comprising a modified oligonucleotide of the formula Tes Geo Teo Aeo mCeo Teo Tds mCds Ads
mCds Ads Tds Tds Tds Gds Gds Aeo Ges mCes mCe (SEQ ID NO: 1185), wherein
A = adenine nucleobase,
mC = 5 -methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
Said oligomeric compound, wherein s = phosphorothioate internucleoside linkages and o = phosphodiester internucleoside linkages.

Embodiment 70. An oligomeric compound comprising a modified oligonucleotide of the formula Tes Geo Geo Aeo Teo Teo mCds Tds Gds Tds Ads mCds Tds Tds Tds Tds Tds mCeo Tes mCes Ae (SEQ ID NO: 3235), wherein
A = adenine nucleobase,
mC = 5 -methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
Said oligomeric compound, wherein s = phosphorothioate internucleoside linkages and o = phosphodiester internucleoside linkages.

Embodiment 71. An oligomeric compound comprising a modified oligonucleotide of the formula: mCes mCeo Teo Aeo Teo mCeo Ads Tds mCds Ads Tds Tds Tds Tds mCds mCds Aeo Ges
Ges Ge (SEQ ID NO: 158), where
A = adenine nucleobase,
mC = 5 -methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
Said oligomeric compound, wherein s = phosphorothioate internucleoside linkages and o = phosphodiester internucleoside linkages.

Embodiment 72. An oligomeric compound comprising a modified oligonucleotide of the formula Tes mCeo Teo Geo Tes Ads mCds Tds Tds
Tds Tds mCds Tds mCds Ads Teo Geo Tes Ges mCe (SEQ ID NO: 2544), wherein
A = adenine nucleobase,
mC = 5 -methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
Said oligomeric compound, wherein s = phosphorothioate internucleoside linkages and o = phosphodiester internucleoside linkages.

Embodiment 73. The oligomeric compound of embodiment 3, wherein said modified oligonucleotide is an RNAi compound.

Embodiment 74. 74. The oligomeric compound of embodiment 73, wherein said RNAi compound is ssRNA or siRNA.

I. Certain Oligonucleotides In certain embodiments, oligomeric compounds are provided herein that comprise an oligonucleotide consisting of linked nucleosides. Oligonucleotides can be unmodified oligonucleotides (RNA or DNA) or can be modified oligonucleotides. Modified oligonucleotides contain at least one modification relative to unmodified RNA or DNA. That is, modified oligonucleotides comprise at least one modified nucleoside (including modified sugar moieties and/or modified nucleobases) and/or at least one modified internucleoside linkage.

A. Certain Modified Nucleosides Modified nucleosides include modified sugar moieties or modified nucleobases or both modified sugar moieties and modified nucleobases.

1. Certain Sugar Moieties In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may contain one or more substitutions corresponding to other types of modified sugar moieties.

In certain embodiments, the modified sugar moiety is non-bicyclic comprising a furanosyl ring having one or more substituents, none of which bridge two atoms of the furanosyl ring to form a bicyclic structure. is a modified sugar moiety of Such non-bridging substituents can be at any position of the furanosyl including, but not limited to, the 2', 4', and/or 5' positions. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moieties are branched. Examples of suitable 2′-substituent groups for non-bicyclic modified sugar moieties include, but are not limited to, 2′-F, 2′-OCH ₃ (“OMe” or “O-methyl” ), and 2′-O(CH ₂ ) ₂ OCH ₃ (“MOE”). In certain embodiments, the 2′-substituent is halo, allyl, amino, azido, SH, CN, OCN, CF ₃ , OCF ₃ , O—C ₁ -C ₁₀ alkoxy, O—C ₁ -C ₁₀ substituted alkoxy, O—C ₁ -C ₁₀ alkyl, O—C ₁ -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(CH ₂ ) ₂ SCH ₃ , O (CH ₂ ) ₂ ON(R _m )(R _n ), or OCH ₂ C(═O)—N(R _m )(R _n ), each R _m and R _n independently H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl, the 2′-substituent being as described in Cook et al. , U. S. 6,531,584, Cook et al. , U. S. 5,859,221 and Cook et al. , U. S. 6,005,087. Certain embodiments of these 2'-substituents are hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro ₍ NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. can be further substituted with one or more substituents independently selected from Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (eg, methoxy), alkyl, and Manoharan et al. , WO2015/106128. Examples of suitable 5'-substituent groups for non-bicyclic modified sugar moieties include, but are not limited to, 5-methyl (R or S), 5'-vinyl, and 5'-methoxy. mentioned. In certain embodiments, the non-bicyclic modified sugar moieties include two or more non-bridging sugar substituents, eg, 2′-F-5′-methyl sugar moieties, and Migawa et al. , WO2008/101157 and Rajeev et al. , US2013/0203836, including modified sugar moieties and modified nucleosides.

In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside is F _{, NH2} , N3, _{OCF3 ,} OCH3, O( _CH2 ) _3NH2 , _{CH2CH =} CH ₂ , OCH2CH= _CH2 , _OCH2CH2OCH3 , O( _CH2 ) _{2SCH3 ,} O _{( CH2} ) _2ON ( _Rm )( _Rn ), O _{( CH2} ) _{2O (} CH ₂ ) a sugar moiety comprising a non-bridging 2′-substituent selected from ₂ N(CH ₃ ) ₂ , and N-substituted acetamides (OCH ₂ C(═O)—N(R _m )(R _n )); and each R _m and R _n is independently H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

In certain embodiments, the 2′-substituted nucleoside non-bicyclic modified nucleoside is F, OCF ₃ , OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) from 2ON( _CH3 ) ₂ , O( _CH2 ) _2O ( _CH2 )2N( _CH3 ) ₂ , and _{OCH2C ( =} O)-N(H) _CH3 ("NMA"); Includes sugar moieties with selected non-bridging 2'-substituents.

In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside has a sugar moiety comprising a non _- bridging 2' _- substituent selected from F, OCH3 _, and _OCH2CH2OCH3 . include.

Certain modified sugar moieties include substituents that bridge two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to, 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) _3-2 ′, 4′-CH ₂ -O-2′ (“LNA”), 4′-CH ₂ -S-2′, 4′-(CH ₂ ) ₂ -O-2′ (“ENA”) ), 4′-CH(CH ₃ )-O-2′ (referred to as “constrained ethyl” or “cEt”), 4′-CH ₂ —O—CH ₂ -2′, 4′-CH ₂ — N(R)-2′,4′-CH(CH ₂ OCH ₃ )-O-2′ (“constrained MOE” or “cMOE”) and analogs thereof (eg, Seth et al., US 7 , 399,845, Bhat et al., U.S. 7,569,686, Swayze et al., U.S. 7,741,457, and Swayze et al., U.S. 8,022,193. ), 4′-C(CH ₃ )(CH ₃ )—O-2′ and analogues thereof (see, eg, Seth et al., US 8,278,283), 4′-CH ₂ -N(OCH3)-2' and analogues thereof (see, e.g., Prakash et al., US 8,278,425), 4'- _{CH2 -} ON( _CH3 )-2 ' (see, e.g., Allerson et al., US 7,696,345 and Allerson et al., US 8,124,745), 4'- _CH2 -C(H)( _CH3 )-2′ (see, for example, Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4′-CH ₂ —C(═CH ₂ )-2′ and analogues thereof (See, eg, Seth et al., US 8,278,426), 4′-C(R _a R _b )—N(R)—O-2′, 4′-C(R _a R _b ) —ON(R)-2′, 4′-CH ₂ —ON(R)-2′, and 4′-CH ₂ —N(R)-O-2′, wherein each R, R _a , and R _b are independently H, protecting groups, or C ₁ -C ₁₂ alkyl (see, eg, Imanishi et al., US 7,427,672).

In certain embodiments, such 4′ to 2′ bridges are independently —[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) 1 to 4 linked groups independently selected from -, -O-, -Si(R _a ) ₂ -, -S(=O) _x -, and -N(R _a )- ,
During the ceremony,
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
Each R _a and R _b is independently H, protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C _{2 —C12 alkynyl} , substituted _{C2 -} C12 alkynyl, C5 _{- C20} aryl, substituted C5 _{- C20} aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5 _- C7 aliphatic cyclic radicals, substituted C ₅ -C ₇ alicyclic radicals, halogens, OJ ₁ , NJ ₁ J ₂ , SJ ₁ , N ₃ , COOJ ₁ , acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) ₂ -J ₁ ), or sulfoxyl (S(=O)-J ₁ );
Each J ₁ and J ₂ is independently H, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ -C ₁₂ aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art and can be found, for example, in 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. , 19
98, 4, 455-456, Koshkin et al. , Tetrahedron, 1998, 54, 3607-3630, Kumar et al. , Bioorg. Med. Chem. Lett. , 1998, 8, 2219-2222, Singh et al. , J. Org. Chem. , 1998, 63, 10035-10039, Srivastava et al. , J. Am. Chem. Soc. , 2007, 129, 8362-8379, Wengel et al. , U. S. 7,053,207, Imanishi et al. , U. S. 6,268,490, Imanishi et al. U.S.A. S. 6,770,748, Imanishi et al. , U. S. RE 44,779, Wengel et al. , U. S. 6,794,499, Wengel et al. , U. S. 6,670,461, Wengel et al. , U. S. 7,034,133, Wengel et al. , U. S. 8,080,644, Wengel et al. , U. S. 8,034,909, Wengel et al. , U. S. 8, 153, 365, Wengel et al. , U. S. 7,572,582, and Ramasamy et al. , U. S. 6,525,191, Torsten et al. , WO2004/106356, Wengel et al. , WO 1999/014226, Seth et al. , WO2007/134181, Seth et al. , U. S. 7,547,684, Seth et al. , U. S. 7,666,854, Seth et al. , U. S. 8,088,746, Seth et al. , U. S. 7,750,131, Seth et al. , U. S. 8,030,467, Seth et al. , U. S. 8,268,980, Seth et al. , U. S. 8,546,556, Seth et al. , U. S. 8,530,640, Migawa et al. , U. S. 9,012,421, Seth et al. , U. S. 8,501,805, and US Patent Publication Allerson et al. , US 2008/0039618 and Migawa et al. , US 2015/0191727.

α－Ｌ－メチレンオキシ（４’－ＣＨ_２－Ｏ－２’）またはα－Ｌ－ＬＮＡ二環式ヌクレオシドは、アンチセンス活性を示したオリゴヌクレオチドに抱合されている（Ｆｒｉｅｄｅｎ ｅｔ ａｌ．，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，２００３，２１，６３６５－６３７２）。本明細書において、二環式ヌクレオシドの一般的な説明は、両方の異性体配置を含む。特定の二環式ヌクレオシド（例えば、ＬＮＡまたはｃＥｔ）の位置が本明細書に例示される実施形態で特定される場合、別段の定めがない限り、それらはβ－Ｄ配置にある。 In certain embodiments, bicyclic sugar moieties and nucleosides that conjugate such bicyclic sugar moieties are further defined by isomeric configuration. For example, LNA nucleosides (described herein) can be in the α-L or β-D configuration.

α-L-methyleneoxy (4′-CH ₂ -O-2′) or α-L-LNA bicyclic nucleosides have been conjugated to oligonucleotides that have shown antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this specification the general description of bicyclic nucleosides includes both isomeric configurations. Where certain bicyclic nucleoside (eg, LNA or cEt) positions are specified in the embodiments illustrated herein, they are in the β-D configuration unless otherwise specified.

In certain embodiments, modified sugar moieties include one or more non-bridging sugar substituents and one or more bridging sugar substituents (eg, 5′-substituted and 4′-2′-bridged sugars). .

In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, oxygen atoms of the sugar moiety are replaced with, for example, sulfur, carbon, or nitrogen atoms. In certain such embodiments, such modified sugar moieties also include bridging and/or non-bridging substituents, as described herein. For example, certain sugar surrogates have a 4′-sulfur atom as well as a 2′-position (eg, Bhat et al., US 7,875,733 and Bhat et al., US 7,939, 677) and/or substitutions at the 5' position.

（「Ｆ－ＨＮＡ」、例えば、Ｓｗａｙｚｅ ｅｔ ａｌ．，Ｕ．Ｓ．８，０８８，９０４、Ｓｗａｙｚｅ ｅｔ ａｌ．，Ｕ．Ｓ．８，４４０，８０３、Ｓｗａｙｚｅ ｅｔ ａｌ．，Ｕ．Ｓ．８，７９６，４３７、及びＳｗａｙｚｅ ｅｔ ａｌ．，Ｕ．Ｓ．９，００５，９０６を参照；Ｆ－ＨＮＡはまた、Ｆ－ＴＨＰまたは３’－フルオロテトラヒドロピランとも称され得る）、及び以下の式を有する追加の修飾されたＴＨＰ化合物を含むヌクレオシドを含み、

式中、独立して、当該修飾されたＴＨＰヌクレオシドの各々について、
Ｂｘは、核酸塩基部分であり、
Ｔ_３及びＴ_４は各々独立して、修飾されたＴＨＰヌクレオシドをオリゴヌクレオチドの残りに結合するヌクレオシド間結合基であるか、またはＴ_３及びＴ_４の一方は、修飾されたＴＨＰヌクレオシドをオリゴヌクレオチドの残りに結合するヌクレオシド間結合基であり、Ｔ_３及びＴ_４の他方は、Ｈ、ヒドロキシル保護基、結合されたコンジュゲート基、または５’もしくは３’－末端基であり、
ｑ_１、ｑ_２、ｑ_３、ｑ_４、ｑ_５、ｑ_６、及びｑ_７はそれぞれ独立して、Ｈ、Ｃ_１－Ｃ_６アルキル、置換Ｃ_１－Ｃ_６アルキル、Ｃ_２－Ｃ_６アルケニル、置換Ｃ_２－Ｃ_６アルケニル、Ｃ_２－Ｃ_６アルキニル、または置換Ｃ_２－Ｃ_６アルキニルであり、
Ｒ_１及びＲ_２のそれぞれは独立して、水素、ハロゲン、置換または非置換アルコキシ、ＮＪ_１Ｊ_２、ＳＪ_１、Ｎ_３、ＯＣ（＝Ｘ）Ｊ_１、ＯＣ（＝Ｘ）ＮＪ_１Ｊ_２、ＮＪ_３Ｃ（＝Ｘ）ＮＪ_１Ｊ_２、及びＣＮの中から選択され、Ｘは、Ｏ、Ｓ、またはＮＪ_１であり、各Ｊ_１、Ｊ_２、及びＪ_３は独立して、ＨまたはＣ_１－Ｃ_６アルキルである。 In certain embodiments, sugar surrogates include rings with more than 5 atoms. For example, in certain embodiments the sugar surrogate comprises a 6-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides, including such modified tetrahydropyrans, include, but are not limited to, hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), mannitol nucleic acid (“MNA”) (e.g., Leumann, CJ. Bioorg). & Med. Chem. 2002, 10, 841-854), fluoro HNA:

(“F-HNA”, eg, Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S. 8, 796, 437, and Swayze et al., U.S. 9,005, 906; F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran), and has the formula a nucleoside comprising an additional modified THP compound;

wherein independently for each of said modified THP nucleosides:
Bx is a nucleobase moiety;
T3 and _{T4 are each} independently an internucleoside linking group that links the modified THP nucleoside to the rest of the oligonucleotide _, or one of T3 and T4 links the modified THP nucleoside to the rest of the oligonucleotide. and the other of T ₃ and T ₄ is H, a hydroxyl protecting group, an attached conjugate group , or a 5′ or 3′-terminal group;
q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ are each independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl , substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, or substituted C ₂ -C ₆ alkynyl,
each of R ₁ and R ₂ is independently hydrogen, halogen, substituted or unsubstituted alkoxy, NJ ₁ J ₂ , SJ ₁ , N ₃ , OC(=X)J ₁ , OC(=X)NJ ₁ J ₂ , NJ ₃ C(=X) NJ ₁ J ₂ , and CN, where X is O, S, or NJ ₁ and each J ₁ , J ₂ , and J ₃ is independently H or C ₁ -C ₆ alkyl.

In certain embodiments, modified THP nucleosides are provided wherein q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ are each H. In certain embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ is other than H. In certain embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R ₁ and R ₂ is F. In certain embodiments R ₁ is F and R ₂ is H; in certain embodiments R ₁ is methoxy and R ₂ is H; In a form R ₁ is methoxyethoxy and R ₂ is H.

In certain embodiments, sugar surrogates include rings with more than 5 atoms and 2 or more heteroatoms. For example, their use in nucleosides and oligonucleotides containing morpholino sugar moieties has been reported (see, eg, Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., US 5, 698). , 685, Summerton et al., U.S. 5,166,315, Summerton et al., U.S. 5,185,444, and Summerton et al., U.S. 5,034,506. ). As used herein, "morpholino" means a sugar surrogate having the structure:

In certain embodiments, morpholinos can be modified, for example, by adding or altering various substituents from the morpholino structures described above. Such sugar surrogates are referred to herein as "modified morpholinos."

In certain embodiments, a sugar surrogate comprises an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar surrogates include, but are not limited to, peptide nucleic acids (“PNA”), acyclic butyl nucleic acids (eg, Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and Manoharan et al. , WO2011/133876.

Many other bicyclic and tricyclic sugars and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

2. Certain Modified Nucleobases In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising unmodified nucleobases. In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides that do not contain a nucleobase, referred to as abasic nucleosides.

In certain embodiments, modified nucleobases are 5-substituted pyrimidines, 6-azapyrimidines, alkyl- or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-
2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobases are 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—CH ₃ )uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5- Ribociluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, especially 5-bromo , 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3 - deazaadenine, 6-N-benzoyl adenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N- It is selected from benzoyluracil, common bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases. Further modified nucleobases include 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxy Including tricyclic pyrimidines such as sazin-2-one (G-clamp). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases are described by Merigan et al. , U. S. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. Am. I. , Ed. , John Wiley & Sons, 1990, 858-859, Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613; S. , Chapter 15, Antisense Research and Applications, Crooke, S.; T. and Lebleu, B.; , Eds. , CRC Press, 1993, 273-288, and Chapters 6 and 15, Antisense Drug Technology, Crooke S.; T. , Ed. , CRC Press, 2008, 163-166 and 442-443.

Publications teaching the preparation of certain of the above modified nucleobases, as well as other modified nucleobases, include, without limitation, Manohara et al. , US2003/0158403, Manoharan et al. , US2003/0175906, Dinh et al. , U. S. 4,845,205, Spielvogel et al. , U. S. 5,130,302, Rogers et al. , U. S. 5,134,066, Bischofberger et al. , U. S. 5, 175, 273, Urdea et al. , U. S. 5,367,066, Benner et al. , U. S. 5,432,272, Matteucci et al. , U. S. 5,434,257, Gmeiner et al. , U. S. 5,457,187, Cooket
al. , U. S. 5,459,255, Froehler et al. , U. S. 5,484,908, Matteucci et al. , U. S. 5,502,177, Hawkins et al. , U. S. 5,525,711, Haralambidis et al. , U. S. 5,552,540, Cook et al. , U. S. 5,587,469, Froehler et al. , U. S. 5,594,121, Switzer et al. , U. S. 5,596,091, Cook et al. , U. S. 5,614,617, Froehler et al. , U. S. 5,645,985, Cook et al. , U. S. 5,681,941, Cook et al. , U. S. 5,811,534, Cook et al. , U. S. 5,750,69
2, Cook et al. , U. S. 5,948,903, Cook et al. , U. S. 5,587,470, Cook et al. , U. S. 5,457,191, Matteucci et al. , U. S. 5,763,588, Froehler et al. , U. S. 5,830,653, Cook et al. , U. S. 5,808,027, Cook et al. , 6, 166, 199, and Matteucci et al. , U. S. Including 6,005,096.

3. Certain Modified Internucleoside Linkages In certain embodiments, the nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates, phosphotriesters, methylphosphonates, including phosphodiester linkages ("P=O") (also referred to as unmodified or native linkages). , phosphoramidates, and phosphorothioates (“P=S”), and phosphorodithioates (“HS-P=S”). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH ₂ --N(CH ₃ )--O--CH ₂ --), thiodiesters, thionocarbamates (--O-- C(=O)(NH)-S-), siloxane (-O-SiH ₂ -O-), and N,N'-dimethylhydrazine (-CH ₂ -N(CH ₃ )-N(CH ₃ )- ) is included. Modified internucleoside linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to native phosphate linkages. In certain embodiments, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Methods of preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

特に指示がない限り、本明細書に記載される修飾されたオリゴヌクレオチドのキラルヌクレオシド間結合は、ステレオランダムであり得るか、または特定の立体化学的配置にあり得る。 Representative internucleoside linkages with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides containing internucleoside linkages with chiral centers can be in specific stereochemical configurations as modified oligonucleotides containing stereorandom internucleoside linkages or as populations of modified oligonucleotides containing phosphorothioate linkages. can be prepared as In certain embodiments, the population of modified oligonucleotides comprises phosphorothioate internucleoside linkages, and all phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be produced using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nevertheless, as well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined configuration. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides comprising one or more specific phosphorothioate internucleoside linkages in specific, independently selected stereochemical configurations. ing. In certain embodiments, a particular configuration of a particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be prepared by synthetic methods known in the art, eg, Oka et al. , JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO2017/015555. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioates each comprise one or more of the following formulae, where "B" represents a nucleobase.

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

Neutral internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3′-CH ₂ —N(CH ₃ )—O-5′), amide-3 (3′-CH ₂ — C(=O)-N(H)-5'), amide-4(3'- _CH2 -N(H)-C(=O)-5'), formacetal (3'-O- _CH2 -O-5'), methoxypropyl, and thioformacetal (3'-S-CH ₂ -O-5'). Additional neutral internucleoside linkages include nonionic linkages including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, for example, Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and _CH2 component moieties.

B. Certain Motifs In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising modified sugar moieties. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkages. In such embodiments, modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of modified oligonucleotides define a pattern or motif. In certain embodiments, the sugar moieties, nucleobases, and internucleoside linkage patterns are each independent of each other. Thus, modified oligonucleotides can be described by their sugar motifs, nucleobase motifs, and/or internucleoside linkage motifs (as used herein, nucleobase motifs are independent of the sequence of the nucleobases). describes modifications to the nucleobases that are

1. Certain Sugar Motifs In certain embodiments, the oligonucleotide has one or more types of modified sugars and/or modified sugars arranged along the oligonucleotide or regions thereof in a defined pattern or sugar motif. contains no sugar moieties. In certain examples, such sugar motifs include, but are not limited to, any of the sugar modifications described herein.

In certain embodiments, the modified oligonucleotide comprises or consists of a region with a gapmer motif defined by two outer regions or "wings" and a central or inner region or "gap". The three regions of the gapmer motif (5'-wing, gap, and 3'-wing) form a continuous sequence of nucleosides, and at least a portion of the sugar moieties of each nucleoside of the wing are aligned with the nucleosides of the gap. different from at least a portion of the sugar moieties. Specifically, at least the sugar moieties of the nucleosides of each wing most adjacent to the gap (the 5'-most nucleoside of the wing and the 5'-most nucleoside of the 3'-wing (5'- The most nucleoside) is distinct from the sugar moieties of the adjacent gap nucleosides and thus defines the boundary between the wing and the gap (ie the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as each other. In certain embodiments, a gap includes one or more nucleosides that have sugar moieties that differ from the sugar moieties of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric gapmers). In certain embodiments, the 5'-wing sugar motif is different from the 3'-wing sugar motif (asymmetric gapmer).

In certain embodiments, the gapmer wings comprise 1-5 nucleosides. In certain embodiments, each nucleoside of each wing of the gapmer is a modified nucleoside. In certain embodiments, at least one nucleoside of each wing of the gapmer is a modified nucleoside. In certain embodiments, at least two nucleosides of each wing of the gapmer are modified nucleosides. In certain embodiments, at least three nucleosides of each wing of the gapmer are modified nucleosides. In certain embodiments, at least four nucleosides of each wing of the gapmer are modified nucleosides.

In certain embodiments, gapmer gaps contain 7-12 nucleosides. In certain embodiments, each nucleoside of a gapmer gap is an unmodified 2'-deoxynucleoside. In certain embodiments, at least one nucleoside in a gapmer gap is a modified nucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In certain embodiments, the gap side nucleoside of each wing/gap junction is an unmodified 2'-deoxynucleoside and the wing side nucleoside of each wing/gap junction is a modified nucleoside. In certain embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In certain embodiments, each nucleoside of each wing of the gapmer is a modified nucleoside.

In certain embodiments, a modified oligonucleotide comprises or consists of a region with a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, each nucleoside throughout the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif, wherein each nucleoside within the fully modified region is uniformly containing the same modified sugar moieties, referred to as modified sugar motifs. In certain embodiments, fully modified oligonucleotides are uniformly modified oligonucleotides. In certain embodiments, each nucleoside of a uniformly modified one contains the same 2'-modification.

As used herein, the lengths (number of nucleosides) of the three regions of the gapmer are represented by the notation [number of nucleosides in the 5′-wing]−[number of nucleosides in the gap]−[number of nucleosides in the 3′-wing number]. Thus, a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap. When such terminology is followed by a specific modification, that modification is a modification at each sugar moiety of each wing, and gap nucleosides include unmodified deoxynucleoside sugars. Thus, a 5-10-5 MOE gapmer has 5 linked MOE modified nucleosides in the 5'-wing, 10 deoxynucleosides in the gap, and 5 linked MOE nucleosides in the 3'-wing. consists of

In certain embodiments, modified oligonucleotides are 5-10-5 MOE gapmers. In certain embodiments the modified oligonucleotide is a 3-10-3BNA gapmer. In certain embodiments the modified oligonucleotide is a 3-10-3cEt gapmer. In certain embodiments the modified oligonucleotide is a 3-10-3 LNA gapmer.

2. Certain Nucleobase Motifs In certain embodiments, an oligonucleotide comprises modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments some or all of the cytosine nucleobases in the modified oligonucleotide are 5-methylcytosine. In certain embodiments, all of the cytosine nucleobases are 5-methylcytosine and all other nucleobases of the modified oligonucleotide are unmodified nucleobases.

In certain embodiments, modified oligonucleotides comprise blocks of modified nucleobases. In certain such embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 3'-end of the oligonucleotide. In certain embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 5'-end of the oligonucleotide.

In certain embodiments, oligonucleotides with gapmer motifs comprise nucleosides comprising modified nucleobases. In certain such embodiments, one nucleoside comprising the modified nucleobase is in the central gap of the oligonucleotide with the gapmer motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2'-deoxyribosyl moiety. In certain embodiments, modified nucleobases are selected from 2-thiopyrimidines and 5-propynepyrimidines.

3. Certain Internucleoside Linkage Motifs In certain embodiments, the oligonucleotide comprises modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or regions thereof in a defined pattern or motif. include. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage (P=O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate internucleoside linkage (P=S). In certain embodiments, each internucleoside linkage of the modified oligonucleotide is independently selected from phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, each phosphorothioate internucleoside linkage is independently selected from stereorandom phosphorothioates, (Sp) phosphorothioates, and (Rp) phosphorothioates. In certain embodiments, the sugar motifs of the modified oligonucleotide are gapmers and all internucleoside linkages within the gap are modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphodiester internucleoside linkages. In certain embodiments, terminal internucleoside linkages are modified. In certain embodiments, the sugar motif of the modified oligonucleotide is a gapmer, the internucleoside linkage motif comprises at least one phosphodiester internucleoside linkage in at least one wing, and at least one phosphodiester linkage is not a terminal internucleoside linkage and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. In certain such embodiments, all of the phosphorothioate linkages are stereorandom. In certain embodiments, all phosphorothioate linkages in the wings are (Sp) phosphorothioate and the gap contains at least one Sp, Sp, Rp motif. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides comprising such internucleoside linkage motifs.

C. Certain Lengths It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992) on their ability to induce target RNA cleavage in an oocyte injection model. tested. A 25 nucleobase long oligonucleotide with 8 or 11 mismatched bases near the end of the oligonucleotide can direct specific cleavage of a target mRNA, albeit to a lesser extent than an oligonucleotide without mismatches. rice field. Similarly, target-specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.

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

D. Certain Modified Oligonucleotides In certain embodiments, the above modifications (sugars, nucleobases, internucleoside linkages) are conjugated to modified oligonucleotides. In certain embodiments, modified oligonucleotides are characterized by their modification, motif, and overall length. In certain embodiments, each such parameter is independent of the other. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may or may not be modified, and may or may not follow the gapmer modification pattern of sugar modifications. good too. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from each other and may be the same or different from the internucleoside linkages of the gap regions of the sugar motif. Similarly, such sugar gapmer oligonucleotides may contain one or more modified nucleobases independent of the gapmer pattern of sugar modifications. All modifications are independent of the nucleobase sequence unless otherwise indicated.

E. Certain Populations of Modified Oligonucleotides A population of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be a stereorandom population or a chirally enriched population. All chiral centers of the modified oligonucleotides are stereorandom in the stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides of the population. In certain embodiments, the chirally enriched population of modified oligonucleotides are enriched in β-D ribosyl sugar moieties and all of the phosphorothioate internucleoside linkages are stereorandom. In certain embodiments, the chirally enriched population of modified oligonucleotides has both a β-D ribosyl sugar moiety and at least one specific phosphorothioate internucleoside linkage in a specific stereochemical configuration. is concentrated.

F. Nucleobase Sequence In certain embodiments, oligonucleotides (unmodified or modified) are further described by their nucleobase sequence. In certain embodiments, an oligonucleotide has a nucleobase sequence complementary to an identified reference nucleic acid, such as a second oligonucleotide or target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence complementary to an identified reference nucleic acid, such as a second oligonucleotide or target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, a nucleic acid such as a second oligonucleotide or target nucleic acid, At least 85%, at least 90%, at least 95%, or 100% complementary.

II. Certain Oligomeric Compounds In certain embodiments, provided herein are oligomeric compounds consisting of oligonucleotides (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. be done. A conjugate group consists of one or more conjugate moieties and a conjugate linker that joins the conjugate moieties to the oligonucleotide. Conjugate groups can be attached to either or both termini of the oligonucleotide and/or at any internal position. In certain embodiments, a conjugate group can be attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, the conjugate groups attached to either or both ends of the oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3' and/or 5'-end of the oligonucleotide. In certain such embodiments, a conjugate group (or terminal group) is attached at the 3'-end of the oligonucleotide. In certain embodiments, the conjugate group is attached proximally to the 3'-end of the oligonucleotide. In certain embodiments, a conjugate group (or terminal group) is attached at the 5'-end of the oligonucleotide. In certain embodiments, the conjugate group is attached proximally to the 5'-end of the oligonucleotide.

Examples of terminal groups include, but are not limited to, conjugate groups , capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and independently modified or unmodified two The above nucleosides can be mentioned.

A. Certain conjugate groups
In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups . In certain embodiments, the conjugate group is a conjugated group, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cell distribution, cell uptake, charge, and clearance. modify one or more properties of the oligonucleotide. In certain embodiments, the conjugate group imparts new properties to the attached oligonucleotide, eg, a fluorophore or reporter group that allows detection of the oligonucleotide. Certain conjugate groups and moieties have been previously described, 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. NY Acad. Sci. , 1992, 660, 306-309, Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. 533-538), aliphatic chains such as do-decane-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118, Kabanov et al., FEBS Lett., 1990 , 259, 327-330, Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac - glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654, Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or palmityl adamantane acetate moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylamino- Carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol groups (Nishina et al., Molecular Ther apy Nucleic Acids, 2015, 4, e220, and Nishina et al. , Molecular Therapy, 2008, 16, 734-740), or the GalNAc cluster (eg WO2014/179620).

1. Conjugate part
Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folates, lipids, Phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and dyes.

In certain embodiments, the conjugate moiety is an active drug substance such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antimicrobials, or antibiotics Contains substances.

2. conjugate linker
A conjugate moiety is attached to the oligonucleotide by a conjugate linker. In certain oligomeric compounds, the conjugate linker is a single chemical bond (ie, the conjugate moiety is directly attached to the oligonucleotide by a single bond). In certain embodiments, the conjugate linker comprises chain structures such as hydrocarbyl chains or oligomers of repeating units such as ethylene glycol, nucleoside, or amino acid units.

In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amido, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amido, and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amido groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, a conjugate linker comprises at least one phosphorus moiety. In certain embodiments, a conjugate linker comprises at least one phosphate group. In certain embodiments, a conjugate linker comprises at least one neutral binding group.

In certain embodiments, conjugate linkers, including the conjugate linkers described above, link a bifunctional linking moiety, e.g., a conjugate group , to a parent compound, such as an oligonucleotide provided herein. are known in the art to be useful for Generally, a bifunctional linking moiety includes at least two functional groups. One of the functional groups is selected to react with a specific site on the parent compound and the other is selected to react with the conjugate group . Examples of functional groups used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophiles and nucleophiles for reacting with electrophiles. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) , and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, or substituted or unsubstituted C ₂ -C ₁₀ alkynyl. , a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain embodiments, a conjugate linker comprises 1-10 linker nucleosides. In certain embodiments, a conjugate linker comprises 2-5 linker nucleosides. In certain embodiments, a conjugate linker comprises exactly 3 linker nucleosides. In certain embodiments, a conjugate linker comprises a TCA motif. In certain embodiments such linker nucleosides are modified nucleosides. In certain embodiments, such linker nucleosides contain modified sugar moieties. In certain embodiments, linker nucleosides are unmodified. In certain embodiments, the linker nucleoside comprises an optionally protected heterocyclic base selected from purines, substituted purines, pyrimidines, or substituted pyrimidines. In certain embodiments, the cleavable moiety is uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenin , guanine, and 2-N-isobutyrylguanine. It is typically desired that the linker nucleoside is cleaved from the oligomeric compound after reaching the target tissue. Thus, the linker nucleosides are typically linked to each other and to the remainder of the oligomeric compound by cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

Linker nucleosides are not considered part of the oligonucleotide herein. Thus, an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percentage of complementarity with a reference nucleic acid, the oligomeric compound also comprising a conjugate linker comprising linker nucleosides. In embodiments that include conjugate groups , those linker nucleosides are not calculated into the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide to the reference nucleic acid. For example, an oligomeric compound can comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker nucleosides flanking the nucleosides of the modified oligonucleotide. can include The total number of adjacent linked nucleosides in such oligomeric compounds exceeds 30. Alternatively, oligomeric compounds may comprise modified oligonucleotides consisting of 8-30 nucleosides and no conjugate groups present. The total number of adjacent linked nucleosides in such oligomeric compounds is 30 or less. Unless otherwise indicated, conjugate linkers contain no more than 10 linker nucleosides. In certain embodiments, a conjugate linker comprises 5 or fewer linker nucleosides. In certain embodiments, a conjugate linker comprises 3 or fewer linker nucleosides. In certain embodiments, a conjugate linker comprises no more than 2 linker nucleosides. In certain embodiments, a conjugate linker includes no more than one linker nucleoside.

In certain embodiments, it is desirable to cleave the conjugate group from the oligonucleotide. For example, in certain circumstances, oligomeric compounds containing certain conjugate moieties are well taken up by certain cell types, but once the oligomeric compound is taken up, the conjugate group is cleaved to render it unconjugated or It is desirable to release the parent oligonucleotide. Accordingly, certain conjugate linkers may contain one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, cleavable moieties include groups of atoms having 1, 2, 3, 4, 5 or more cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or intracellular compartment, such as a lysosome. In certain embodiments, cleavable moieties are selectively cleaved by endogenous enzymes such as nucleases.

In certain embodiments, the cleavable linkage is selected from among amides, esters, ethers, one or both of phosphodiesters, phosphate esters, carbamates, or disulfides. In certain embodiments, the cleavable linkage is one or both esters of a phosphodiester. In certain embodiments, cleavable moieties include phosphates or phosphodiesters. In certain embodiments, the cleavable moiety is a phosphate bond between the oligonucleotide and the conjugate moiety or group .

In certain embodiments, a cleavable moiety comprises or consists of one or more linker nucleosides. In certain such embodiments, one or more linker nucleosides are linked to each other and/or to the remainder of the oligomeric compound by cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, the cleavable moiety is attached to either the 3' or 5'-terminal nucleoside of the oligonucleotide by a phosphate internucleoside linkage and the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. is a 2'-deoxynucleoside that is covalently attached to. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.

B. Certain Terminal Groups In certain embodiments, oligomeric compounds comprise one or more terminal groups. In certain such embodiments, the oligomeric compound comprises a stabilized 5'-phophate. Stabilized 5'-phosphates include 5'-phosphanates, including but not limited to 5'-vinyl phosphonates. In certain embodiments, a terminal group comprises one or more abasic nucleosides and/or reverse nucleosides. In certain embodiments, a terminal group includes one or more 2'-linked nucleosides. In certain such embodiments, the 2'-linked nucleoside is an abasic nucleoside.

III. Oligomeric Duplexes In certain embodiments, the oligomeric compounds described herein comprise oligonucleotides having a nucleobase sequence complementary to the sequence of the target nucleic acid. In certain embodiments, an oligomeric compound pairs with a second oligomeric compound to form an oligomeric duplex. Such oligomeric duplexes comprise a first oligomeric compound having a region complementary to the target nucleic acid, and a second oligomeric compound having a region complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of the oligomeric duplex comprises (1) a modified or unmodified oligonucleotide and optionally a conjugate group ; and (2) a second modified or It comprises or consists of an unmodified oligonucleotide and optionally a conjugate group . Either or both of the oligomeric compounds of the oligomeric duplex may contain a conjugate group . The oligonucleotides of each oligomeric compound of the oligomeric duplex may contain non-complementary overhanging nucleosides.

IV. Antisense Activity In certain embodiments, oligomeric compounds and oligomeric duplexes are capable of hybridizing to a target nucleic acid and provide at least one antisense activity, and such oligomeric compounds and oligomeric duplexes are It is an antisense compound. In certain embodiments, antisense compounds have antisense activity if they reduce or inhibit the amount or activity of the target nucleic acid by 25% or more in standard cellular assays. In certain embodiments, antisense compounds selectively affect one or more target nucleic acids. Such antisense compounds comprise nucleobase sequences that hybridize to one or more target nucleic acids, provide one or more desired antisense activities, and are synthesized in such a way as to provide significant undesired antisense activities. Does not hybridize to one or more non-target nucleic acids, or does not hybridize to one or more non-target nucleic acids.

In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in the recruitment of proteins that cleave the target nucleic acid. For example, certain antisense compounds effect RNase H-mediated cleavage of target nucleic acids. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA of such RNA:DNA duplexes need not be unmodified DNA. In certain embodiments, antisense compounds are described herein that are sufficiently "DNA-like" to induce RNase H activity. In certain embodiments, one or more non-DNA-like nucleosides in gapmer gaps are tolerated.

In certain antisense activities, antisense compounds or portions of antisense compounds are incorporated into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds result in cleavage of target nucleic acids by Argonaute. Antisense compounds incorporated into RISC are RNAi compounds. RNAi compounds can be double-stranded (siRNA) or single-stranded (ssRNA).

In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in the recruitment of proteins that cleave the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in altered splicing of the target nucleic acid. In certain embodiments, hybridization of antisense compounds to target nucleic acids results in inhibition of binding interactions between target nucleic acids and proteins or other nucleic acids. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid.

Antisense activity can be observed directly or indirectly. In certain embodiments, observation or detection of antisense activity is a change in the amount of a target nucleic acid or a protein encoded by such a target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or Including observation or detection of phenotypic changes in animals.

V. Certain Target Nucleic Acids In certain embodiments, an oligomeric compound comprises or consists of an oligonucleotide comprising a region complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from mature mRNAs and pre-mRNAs that contain introns, exons, and untranslated regions. In certain embodiments, the target RNA is mature mRNA. In certain embodiments, the target nucleic acid is pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is an RNA transcript of a retrogene. In certain embodiments, the target nucleic acid is non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from long non-coding RNA, short non-coding RNA, intronic RNA molecules.

A. Complementarity/Mismatch with Target Nucleic Acid Mismatched bases can be introduced without eliminating activity. For example, Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) have 100% complementarity with bcl-2 mRNA and 3 mismatches with bcl-xL mRNA. , demonstrated the ability of oligonucleotides to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide showed potent anti-tumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14-nucleobase oligonucleotides, and 28- and 42-nucleobase oligonucleotides inhibited translation of human DHFR in a rabbit reticulocyte assay. included sequences of 2 or 3 of the tandem oligonucleotides, respectively, as their ability to terminate . Each of the three 14 nucleobase oligonucleotides alone was able to inhibit translation, albeit to a more modest level than the 28 or 42 nucleobase oligonucleotides.

In certain embodiments, oligonucleotides are complementary to the target nucleic acid throughout the 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, an oligonucleotide is at least 80% complementary to a target nucleic acid over the length of the oligonucleotide and includes a region of 100% or complete complementarity to the target nucleic acid. In certain embodiments, regions of perfect complementarity are 6-20, 10-18, or 18-20 nucleobases in length.

In certain embodiments, oligonucleotides contain one or more mismatched nucleobases to the target nucleic acid. In certain embodiments, antisense activity against a target is reduced by such a mismatch, while activity against a non-target is reduced by a greater amount. Thus, in certain embodiments, oligonucleotide selectivity is improved. In certain embodiments, the mismatch is specifically located within the oligonucleotide with the gapmer motif. In certain embodiments, the mismatch is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5'-end of the gap region. In certain embodiments, the mismatch is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3'-end of the gap region. In certain embodiments, the mismatch is at positions 1, 2, 3, or 4 from the 5'-end of the wing region. In certain embodiments, the mismatch is at positions 4, 3, 2, or 1 from the 3'-end of the wing region.

B. ATXN2
In certain embodiments, the oligomeric compound comprises or consists of an oligonucleotide comprising a region complementary to a target nucleic acid, and the target nucleic acid is ATXN2. In certain embodiments, the ATXN2 nucleic acid has the sequence set forth in SEQ ID NO: 1 (GENBANK Accession No. NM_002973.3) and SEQ ID NO: 2 (complement of GENBANK Accession No. NT_009775.17 truncated from nucleotides 2465000-2616000). have.

In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO:1 or SEQ ID NO:2 reduces the amount of ATXN2 mRNA, and in certain embodiments, reduces the amount of ataxin-2 protein. Reduce. In certain embodiments, oligomeric compounds consist of modified oligonucleotides. In certain embodiments, contacting a cell with an oligomeric compound complementary to SEQ ID NO:1 or SEQ ID NO:2 ameliorates one or more symptoms or features of a neurodegenerative disease. In certain embodiments, oligomeric compounds consist of modified oligonucleotides. In certain embodiments, the symptom or characteristic is ataxia, neuropathy, or aggregate formation. In certain embodiments, contacting cells with modified oligonucleotides complementary to SEQ ID NO: 1 or SEQ ID NO: 2 results in improved motor function, reduced neuropathy, and reduced numbers of aggregates. bring. In certain embodiments, oligomeric compounds consist of modified oligonucleotides.

C. Certain Target Nucleic Acids in Certain Tissues In certain embodiments, the oligomeric compound comprises or consists of an oligonucleotide comprising a region complementary to a target nucleic acid, wherein the target nucleic acid is expressed in tissues that In certain embodiments, pharmacologically relevant tissues are cells and tissues comprising the central nervous system (CNS). Such tissues include brain tissues such as cortex, spinal cord, hippocampus, pons, cerebellum, substantia nigra, red nucleus, medulla, thalamus, and dorsal root ganglia.

VI. Certain Pharmaceutical Compositions In certain embodiments, provided herein are pharmaceutical compositions comprising one or more oligomeric compounds. In certain embodiments, each of the one or more oligomeric compounds consists of modified oligonucleotides. In certain embodiments, a pharmaceutical composition includes a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises or consists of a sterile saline solution and one or more oligomeric compounds. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compounds and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, the pharmaceutical composition comprises or consists of one or more oligomeric compounds and phosphate buffered saline (PBS). In certain embodiments, sterile PBS comprises pharmaceutical grade PBS. In certain embodiments, the pharmaceutical composition comprises or consists of one or more oligomeric compounds and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

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

In certain embodiments, pharmaceutical compositions comprise one or more oligomeric compounds and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. .

In certain embodiments, oligomeric compounds may be mixed with pharmaceutically acceptable active and/or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose administered.

In certain embodiments, a pharmaceutical composition comprising an oligomeric compound includes any pharmaceutically acceptable salt of an oligomeric compound, an ester of an oligomeric compound, or a salt of such an ester. In certain embodiments, pharmaceutical compositions comprising oligomeric compounds comprising one or more oligonucleotides provide biologically active metabolites or residues thereof upon administration to animals, including humans. can (directly or indirectly) Thus, for example, the disclosure also contemplates pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioisosteres of oligomeric compounds. Suitable pharmaceutically acceptable salts include, but are not limited to sodium and potassium salts. In certain embodiments, a prodrug comprises one or more conjugate groups attached to an oligonucleotide, where the conjugate groups are cleaved in the body by endogenous nucleases.

Lipid moieties have been used in nucleic acid therapy in a variety of ways. In certain such methods, nucleic acids, such as oligomeric compounds, are introduced into preformed liposomes or lipoplexes made of mixtures of cationic and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed in the absence of neutral lipids. In certain embodiments, the lipid moieties are selected to increase distribution of the pharmaceutical agent to specific cells or tissues. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to muscle tissue.

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 are useful for preparing certain pharmaceutical compositions, including those containing hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the invention to specific tissues or cell types. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

In certain embodiments, the pharmaceutical composition comprises a co-solvent system. Certain such co-solvent systems include, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is 3% w/v benzyl alcohol, 8% w/v non-polar surfactant Polysorbate 80™, and 65 Solution in absolute ethanol containing % w/v polyethylene glycol 300. The proportions of such co-solvent systems can be varied considerably without significantly altering their solubility and toxicity characteristics. Additionally, the identity of the co-solvent components may vary, e.g. other surfactants may be used in place of Polysorbate 80™, the fraction size of polyethylene glycol may vary. Often other biocompatible polymers may replace polyethylene glycol, eg, polyvinylpyrrolidone, and other sugars or polysaccharides may replace dextrose.

In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, pharmaceutical compositions are prepared for administration by injection (eg, 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 such as water or in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. become. In certain embodiments, other ingredients are included (eg, ingredients that aid solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, and the like. Certain pharmaceutical compositions for injection are in unit dosage forms, eg, in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, and liposomes. include.

Under certain conditions, certain compounds disclosed herein act as acids. While such compounds may be depicted or described in protonated (free acid) form, ionized (anion) form, or associated with ionization and cations (salts), aqueous solutions of such compounds are Among such forms exists in a state of equilibrium. For example, the phosphate linkages of oligonucleotides in aqueous solution exist in equilibrium among free acid, anionic, and salt forms. Unless otherwise indicated, the compounds disclosed herein are meant to include all such forms. Moreover, a given oligonucleotide will have several such bonds, each in equilibrium. Oligonucleotides in solution are therefore all present in equilibrium at multiple positions in the form of an ensemble. The term "oligonucleotide" is intended to include all such forms. The structures depicted necessarily show a single form. Nevertheless, unless otherwise indicated, such figures are intended to include corresponding features as well. As used herein, structures depicting free acids of compounds followed by the term "or a salt thereof" reveal all such forms that may be associated with fully or partially protonated/deprotonated/cationic include. In certain examples, one or more specific cations are specified.

In certain embodiments, oligomeric compounds disclosed herein are in an aqueous solution comprising sodium. In certain embodiments, the oligomeric compound is in water soluble containing potassium. In certain embodiments, oligomeric compounds are in artificial CSF. In certain embodiments, oligomeric compounds are in PBS. In certain embodiments, oligomeric compounds are in water. In certain such embodiments, the pH of the solution is adjusted with NaOH and/or HCl to achieve the desired pH.

VII. Certain Compositions 1 . Compound number 874218
Compound No. 874218 may be characterized as a 5-10-5 MOE gapmer having the sequence (5′ to 3′) of GTACTTTTCTCATGTGCGGC (incorporated herein as SEQ ID NO: 1714), nucleosides 1-5 and 16 -20 each (5'-3') contains a 2'-MOE modification, each of nucleosides 6-15 is a 2'-deoxynucleoside, nucleosides 2-3, 3-4, 4-5, Internucleoside linkages between 5-6, 16-17, and 17-18 are phosphodiester internucleoside linkages, nucleosides 1-2, 6-7, 7-8, 8-9, 9-10, 10 The internucleoside linkages between -11, 11-12, 12-13, 13-14, 14-15, 15-16, 18-19, and 19-20 are phosphorothioate internucleoside linkages, and each cytosine is 5-methylcytosine.

Compound No. 780241 may be characterized by the following formula ( chemical notation ) , Ges Teo Aeo mCeo Teo Tds Tds Tds mCds Tds mCds Ads Tds Gds Tds Geo mCeo Ges Ges mCe, wherein:
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkages, and o = phosphodiester internucleoside linkages.

Compound No. 874218 can be represented by the chemical structure below.

In certain embodiments, the sodium salt of Compound No. 874218 can be represented by the chemical structure below.

2. Compound number 1008854
Compound No. 1008854 may be characterized as a 4-10-6 MOE gapmer with the sequence (5′ to 3′) of CTGCTAACTGGTTTGCCCTT (incorporated herein as SEQ ID NO: 1255), comprising nucleosides 1-4 and 15 -20 (5'-3') includes a 2'-MOE modification, each of nucleosides 5-14 is a 2'-deoxynucleoside, nucleosides 2-3, 3-4, 4-5, The internucleoside linkages between 15-16, 16-17, 17-18 are phosphodiester internucleoside linkages, nucleosides 1-2, 5-6, 6-7, 7-8, 8-9, 9- Internucleoside linkages between 10, 10-11, 11-12, 12-13, 13-14, 14-15, 18-19, and 19-20 are phosphorothioate internucleoside linkages, each cytosine having 5 - is methylcytosine.

Compound No. 1008854 can be characterized by the following formula ( chemical notation ) , mCes Teo Geo mCeo Tds Ads Ads mCds Tds Gds Gds Tds Tds Tds Tds Geo mCeo mCeo mCes Tes Te, wherein:
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkages, and o = phosphodiester internucleoside linkages.

Compound No. 1008854 can be represented by the chemical structure below.

In certain embodiments, the sodium salt of Compound No. 1008854 can be represented by the chemical structure below.

3. Compound number 1008862
Compound No. 1008862 may be characterized as a 6-10-4 MOE gapmer with the sequence (5′ to 3′) of TGTACTTCACATTTGGAGCC (incorporated herein as SEQ ID NO: 1185), nucleosides 1-6 and 17 -20 each (5'-3') contains a 2'-MOE modification, each of nucleosides 7-16 is a 2'-deoxynucleoside, nucleosides 2-3, 3-4, 4-5, Internucleoside linkages between 5-6, 6-7, and 17-18 are phosphodiester internucleoside linkages, nucleosides 1-2, 7-8, 8-9, 9-10, 10-11, 11 The internucleoside linkages between -12, 12-13, 13-14, 14-15, 15-16, 16-17, 18-19, and 19-20 are phosphorothioate internucleoside linkages, each cytosine 5-methylcytosine.

Compound No. 1008862 may be characterized by the following formula ( chemical notation ) , Tes Geo Teo Aeo mCeo Teo Tds mCds Ads mCds Ads Tds Tds Tds Tds Gds Gds Aeo Ges mCes mCe, wherein:
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkages, and o = phosphodiester internucleoside linkages.

Compound No. 1008862 can be represented by the chemical structure below.

In certain embodiments, the sodium salt of Compound No. 1008862 can be represented by the chemical structure below.

4. Compound number 1008870
Compound No. 1008870 may be characterized as a 6-10-4 MOE gapmer having the sequence (5′ to 3′) of TGGATTCTGTACTTTTCTCA (incorporated herein as SEQ ID NO: 3235), nucleosides 1-6 and 17 -20 each (5'-3') contains a 2'-MOE modification, each of nucleosides 7-16 is a 2'-deoxynucleoside, nucleosides 2-3, 3-4, 4-5, Internucleoside linkages between 5-6, 6-7, and 17-18 are phosphodiester internucleoside linkages, nucleosides 1-2, 7-8, 8-9, 9-10, 10-11, 11 The internucleoside linkages between -12, 12-13, 13-14, 14-15, 15-16, 16-17, 18-19, and 19-20 are phosphorothioate internucleoside linkages, each cytosine 5-methylcytosine.

Compound No. 1008870 can be characterized by the following formula ( chemical notation ) , Tes Geo Geo Aeo Teo Teo mCds Tds Gds Tds Ads mCds Tds Tds Tds Tds mCeo Tes mCes Ae, wherein:
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkages, and o = phosphodiester internucleoside linkages.

Compound No. 1008870 can be represented by the chemical structure below.

In certain embodiments, the sodium salt of Compound No. 1008870 can be represented by the chemical structure below.

5. Compound number 1008874
Compound No. 1008874 may be characterized as a 6-10-4 MOE gapmer with the sequence (5′ to 3′) of CCTATCATCATTTTCCAGGG (incorporated herein as SEQ ID NO: 158), nucleosides 1-6 and 17 -20 each (5'-3') contains a 2'-MOE modification, each of nucleosides 7-16 is a 2'-deoxynucleoside, nucleosides 2-3, 3-4, 4-5, Internucleoside linkages between 5-6, 6-7, and 17-18 are phosphodiester internucleoside linkages, nucleosides 1-2, 7-8, 8-9, 9-10, 10-11, 11 The internucleoside linkages between -12, 12-13, 13-14, 14-15, 15-16, 16-17, 18-19, and 19-20 are phosphorothioate internucleoside linkages, each cytosine 5-methylcytosine.

Compound No. 1008874 can be characterized by the following formula ( chemical notation ): mCes mCeo Teo Aeo Teo mCeo Ads Tds mCds Ads Tds Tds Tds Tds Tds mCds mCds Aeo Ges Ges Ge, where A = adenine nucleobase ,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkages, and o = phosphodiester internucleoside linkages.

Compound No. 1008874 can be represented by the chemical structure below.

In certain embodiments, the sodium salt of Compound No. 1008874 can be represented by the chemical structure below.

6. Compound number 1008910
Compound No. 1008910 may be characterized as a 5-10-5 MOE gapmer having the sequence (5′ to 3′) of TCGTACTTTTCTCATGTGC (incorporated herein as SEQ ID NO: 2544), nucleosides 1-5 and 16 -20 each (5'-3') contains a 2'-MOE modification, each of nucleosides 6-15 is a 2'-deoxynucleoside, nucleosides 2-3, 3-4, 4-5, The internucleoside linkages between 16-17 and 17-18 are phosphodiester internucleoside linkages, nucleosides 1-2, 5-6, 6-7, 7-8, 8-9, 9-10, 10 The internucleoside linkages between -11, 11-12, 12-13, 13-14, 14-15, 15-16, 18-19, and 19-20 are phosphorothioate internucleoside linkages, and each cytosine is 5-methylcytosine.

Compound No. 1008910 can be characterized by the following formula ( chemical notion): Tes mCeo Teo Geo Tes Ads mCds Tds Tds Tds Tds
mCds Tds mCds Ads Teo Geo Tes Ges mCe, where:
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkages, and o = phosphodiester internucleoside linkages.

Compound No. 1008910 can be represented by the chemical structure below.

In certain embodiments, the sodium salt of Compound No. 1008910 can be represented by the chemical structure below.

VIII. Certain Comparator Compositions In certain embodiments, previously described in WO2015/143246 and Scores et
al. , Nature, 2017, 544(7650):362-366, both of which are incorporated herein by reference, is a comparator compound. Compound No. 564122 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from TGCATAGATTCCATCAAAG (incorporated herein as SEQ ID NO: 67), where each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564127, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. Compound No. 564127 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from CTCTCCATTTTTCTTCACG (incorporated herein as SEQ ID NO: 33), wherein each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564133, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. compound number 564
133 is a 5-10-5 MOE gapmer having the sequence (5′ to 3′) from GCTAACTGGTTTGCCCTTGC (incorporated herein as SEQ ID NO: 32), each cytosine is a 5-methylcytosine, each The internucleoside linkages are phosphorothioate internucleoside linkages, with each of nucleosides 1-5 and 16-20 containing a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564143, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. Compound No. 564143 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from GGAGCTGGAGAACCATGAGC (incorporated herein as SEQ ID NO: 188), wherein each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564150, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. Compound No. 564150 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from CTGGTACAGTTGCTGCTGCT (incorporated herein as SEQ ID NO: 330), where each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564188, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. Compound No. 564188 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from CCCAAAGGGTTAATTAGGAT (incorporated herein as SEQ ID NO: 2901), where each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564210, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. Compound No. 564210 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from CCCATACGCGGTGAATTCTG (incorporated herein as SEQ ID NO: 112), where each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compound 564216, previously described in WO2015/143246 and Scoles, 2017, is a comparator compound. Compound No. 564216 is a 5-10-5 MOE gapmer having the sequence (5' to 3') from GTGGGATACAAATTCTAGGC (incorporated herein as SEQ ID NO: 190), where each cytosine is a 5-methylcytosine. , each internucleoside linkage is a phosphorothioate internucleoside linkage, and each of nucleosides 1-5 and 16-20 contains a 2'-OCH ₂ CH ₂ OCH ₃ group.

In certain embodiments, compounds described herein are superior to compounds described in WO2015/143246 and Scores, 2017 because they exhibit one or more improved properties.

Compound 874218
For example, as provided in Example 13 (below), compound 874218 exhibited a Functional Observational Overall (FOB) score of 1.00 in wild-type mice, whereas comparator compound numbers 564122, 564127, 564133, 564143 , 564150, 564188, 564210, and 564216 each exhibited a FOB score of 7.00. Therefore, compound 874218 is clearly better tolerated than each of comparator compound numbers 564122, 564127, 564133, 564143, 564150, 564188, 564210, and 564216 in this assay.

Compound 1008854
For example, as provided in Example 13 (below), compound 1008854 exhibited a Functional Observational Overall (FOB) score of 1.00 in wild-type mice, whereas comparator compound numbers 564122, 564127, 564133, 564143 , 564150, 564188, 564210, and 564216 each exhibited a FOB score of 7.00. Therefore, compound 1008854 is clearly better tolerated than each of comparator compound numbers 564122, 564127, 564133, 564143, 564150, 564188, 564210, and 564216 in this assay.

Compound 1008862
For example, as provided in Example 13 (below), compound 1008862 exhibited a Functional Observational Global Observation (FOB) score of 2.50 in wild-type mice, whereas comparator compound numbers 564122, 564127, 564133, 564143 , 564150, 564188, 564210, and 564216 each exhibited a FOB score of 7.00. Therefore, compound 1008862 is clearly better tolerated than each of comparator compound numbers 564122, 564127, 564133, 564143, 564150, 564188, 564210, and 564216 in this assay.

Compound 1008870
For example, as provided in Example 13 (below), compound 1008870 exhibited a Functional Observational Global Index (FOB) score of 1.00 in wild-type mice, whereas comparator compound numbers 564122, 564127, 564133, 564143 , 564150, 564188, 564210, and 564216 each exhibited a FOB score of 7.00. Therefore, compound 1008870 is clearly better tolerated than each of comparator compound numbers 564122, 564127, 564133, 564143, 564150, 564188, 564210, and 564216 in this assay.

Compound 1008874
For example, as provided in Example 13 (below), compound 1008874 exhibited a Functional Observational Global Observation (FOB) score of 1.25 in wild-type mice, whereas comparator compound numbers 564122, 564127, 564133, 564143 , 564150, 564188, 564210, and 564216 each exhibited a FOB score of 7.00. Therefore, compound 1008874 is clearly better tolerated than each of comparator compound numbers 564122, 564127, 564133, 564143, 564150, 564188, 564210, and 564216 in this assay.

Compound 1008910
For example, as provided in Example 13 (below), compound 1008910 exhibited a Functional Observational Global Observation (FOB) score of 0.00 in wild-type mice, whereas comparator compound numbers 564122, 564127, 564133, 564143 , 564150, 564188, 564210, and 564216 each exhibited a FOB score of 7.00. Therefore, compound 1008910 is clearly better tolerated than each of comparator compound numbers 564122, 564127, 564133, 564143, 564150, 564188, 564210, and 564216 in this assay.

IX. Certain Hotspot Areas 1 . Nucleobases 2,455 to 2,483 of SEQ ID NO: 1
In certain embodiments, nucleobases 2,455-2,483 of SEQ ID NO:1 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 2,455-2,483 of SEQ ID NO:1. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 2,455-2,483 of SEQ ID NO:1 achieve at least a 67% reduction of ATXN2 RNA in vitro in a standard cellular assay.

2. Nucleobases 4,393 to 4,424 of SEQ ID NO: 1
In certain embodiments, nucleobases 4,393-4,424 of SEQ ID NO:1 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 4,393-4,424 of SEQ ID NO:1. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NO: 1945, 2020, 2316, 2392, 2469, 2546, 2623, 2697, 2926, 3003, 3080, and 3157 are complementary to nucleobases 4,393-4,424 of SEQ ID NO: 1 .

In certain embodiments, modified oligonucleotides complementary to nucleobases 4,393-4,424 of SEQ ID NO: 1 achieve at least a 64% reduction of ATXN2 RNA in vitro in a standard cellular assay.

3. Nucleobases 4,413 to 4,437 of SEQ ID NO: 1
In certain embodiments, nucleobases 4,413-4,437 of SEQ ID NO:1 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 4,413-4,437 of SEQ ID NO:1. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 4,413-4,437 of SEQ ID NO:1 achieve at least a 68% reduction of ATXN2 RNA in vitro in a standard cellular assay.

4. Nucleobases 4,525 to 4,554 of SEQ ID NO:2
In certain embodiments, nucleobases 4,525-4,554 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 4,525-4,554 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs: 1948, 2319, 2549, 2625, 2701, 2777, 2853, 2929, 3006, 3083, and 3160 are complementary to nucleobases 4,525-4,554 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 4,525-4,554 of SEQ ID NO:2 achieve at least a 79% reduction of ATXN2 RNA in vitro in a standard cellular assay.

5. Nucleobases 4,748 to 4,771 of SEQ ID NO:2
In certain embodiments, nucleobases 4,748-4,771 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 4,748-4,771 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOS:2175, 2626, 2702, 2778, and 3161 are complementary to nucleobases 4,748-4,771 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 4,748-4,771 of SEQ ID NO:2 achieve at least 70% reduction of ATXN2 RNA in vitro in a standard cellular assay.

6. Nucleobases 9,927 to 9,954 of SEQ ID NO:2
In certain embodiments, nucleobases 9,927-9,954 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 9,927-9,954 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 9,927-9,954 of SEQ ID NO:2 achieve at least a 62% reduction of ATXN2 RNA in vitro in a standard cellular assay.

7. Nucleobases 10,345 to 10,368 of SEQ ID NO:2
In certain embodiments, nucleobases 10,345-10,368 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 10,345-10,368 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs:2323, 2400, 2477, 2933, and 3011 are complementary to nucleobases 10,345 to 10,368 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 10,345-10,368 of SEQ ID NO:2 achieve at least 87% reduction of ATXN2 RNA in vitro in a standard cellular assay.

8. Nucleobases 17,153 to 17,182 of SEQ ID NO:2
In certain embodiments, nucleobases 17,153-17,182 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 17,153-17,182 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 17,153-17,182 of SEQ ID NO:2 achieve at least a 68% reduction of ATXN2 RNA in vitro in a standard cellular assay.

9. Nucleobases 18,680 to 18,702 of SEQ ID NO:2
In certain embodiments, nucleobases 18,680-18,702 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 18,680-18,702 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs:2256, 2557, 3014, and 3091 are complementary to nucleobases 18,680 to 18,702 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 18,680-18,702 of SEQ ID NO:2 achieve at least a 65% reduction of ATXN2 RNA in vitro in a standard cellular assay.

10. Nucleobases 23,251 to 23,276 of SEQ ID NO:2
In certain embodiments, nucleobases 23,251-23,276 of SEQ ID NO:2 comprise a hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 23,251-23,276 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 23,251-23,276 of SEQ ID NO:2 achieve at least a 64% reduction of ATXN2 RNA in vitro in a standard cellular assay.

11. Nucleobases 28,081 to 28,105 of SEQ ID NO:2
In certain embodiments, nucleobases 28,081-28,105 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 28,081-28,105 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 28,081-28,105 of SEQ ID NO:2 achieve at least a 62% reduction of ATXN2 RNA in vitro in a standard cellular assay.

12. Nucleobases 28,491 to 28,526 of SEQ ID NO:2
In certain embodiments, nucleobases 28,491-28,526 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 28,491-28,526 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 28,491-28,526 of SEQ ID NO:2 achieve at least a 67% reduction of ATXN2 RNA in vitro in a standard cellular assay.

13. Nucleobases 28,885 to 28,912 of SEQ ID NO:2
In certain embodiments, nucleobases 28,885-28,912 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 28,885-28,912 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs:977, 2486, 2563, 2639, 2715, 2791, 3020, 3097, and 3174 are complementary to nucleobases 28,885 to 28,912 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 28,885-28,912 of SEQ ID NO:2 achieve at least 75% reduction of ATXN2 RNA in vitro in a standard cellular assay.

14. Nucleobases 32,328 to 32,352 of SEQ ID NO:2
In certain embodiments, nucleobases 32,328-32,352 of SEQ ID NO:2 comprise a hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 32,328-32,352 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 32,328-32,352 of SEQ ID NO:2 achieve at least a 66% reduction of ATXN2 RNA in vitro in a standard cellular assay.

15. Nucleobases 32,796 to 32,824 of SEQ ID NO:2
In certain embodiments, nucleobases 32,796-32,824 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 32,796-32,824 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 32,796-32,824 of SEQ ID NO:2 achieve at least 72% reduction of ATXN2 RNA in vitro in a standard cellular assay.

16. Nucleobases 32,809 to 32,838 of SEQ ID NO:2
In certain embodiments, nucleobases 32,809-32,838 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 32,809-32,838 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 32,809-32,838 of SEQ ID NO:2 achieve at least 60% reduction of ATXN2 RNA in vitro in a standard cellular assay.

17. Nucleobases 36,308 to 36,334 of SEQ ID NO:2
In certain embodiments, nucleobases 36,308-36,334 of SEQ ID NO:2 comprise a hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 36,308-36,334 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs:1280, 2338, 2415, 2644, 2720, 2796, 2872, and 2948 are complementary to nucleobases 36,308 to 36,334 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 36,308-36,334 of SEQ ID NO:2 achieve at least a 69% reduction of ATXN2 RNA in vitro in a standard cellular assay.

18. Nucleobases 36,845 to 36,872 of SEQ ID NO:2
In certain embodiments, nucleobases 36,845-36,872 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 36,845-36,872 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 36,845-36,872 of SEQ ID NO:2 achieve at least a 63% reduction of ATXN2 RNA in vitro in a standard cellular assay.

19. Nucleobases 49,147 to 49,173 of SEQ ID NO:2
In certain embodiments, nucleobases 49,147-49,173 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 49,147-49,173 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOS:1439, 2575, 2651, 2727, 2803, 3032, 3109, and 3186 are complementary to nucleobases 49,147 to 49,173 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 49,147-49,173 of SEQ ID NO:2 achieve at least a 69% reduction of ATXN2 RNA in vitro in a standard cellular assay.

20. Nucleobases 57,469 to 57,494 of SEQ ID NO:2
In certain embodiments, nucleobases 57,469-57,494 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 57,469-57,494 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 57,469-57,494 of SEQ ID NO:2 achieve at least a 49% reduction of ATXN2 RNA in vitro in a standard cellular assay.

21. Nucleobases 82,848 to 82,874 of SEQ ID NO:2
In certain embodiments, nucleobases 82,848-82,874 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 82,848-82,874 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOS:1982, 2359, 2436, 2513, 2590, 2969, 3047, and 3124 are complementary to nucleobases 82,848 to 82,874 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 82,848-82,874 of SEQ ID NO:2 achieve at least a 57% reduction of ATXN2 RNA in vitro in a standard cellular assay.

22. Nucleobases 83,784 to 83,813 of SEQ ID NO:2
In certain embodiments, nucleobases 83,784-83,813 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 83,784-83,813 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOS:849, 2361, 2438, 2515, 2592, 2668, 2744, 2971, 3049, 3126, and 3203 are complementary to nucleobases 83,784-83,813 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 83,784-83,813 of SEQ ID NO:2 achieve at least 76% reduction of ATXN2 RNA in vitro in a standard cellular assay.

23. Nucleobases 84,743 to 84,782 of SEQ ID NO:2
In certain embodiments, nucleobases 84,743-84,782 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 84,743-84,782 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NO: 2210, 2441, 2518, 2595, 2671, 2747, 2823, 2899, 2975, 3052, 3129, and 3206 are complementary to nucleobases 84,743 to 84,782 of SEQ ID NO:2 .

In certain embodiments, modified oligonucleotides complementary to nucleobases 84,743-84,782 of SEQ ID NO:2 achieve at least a 58% reduction of ATXN2 RNA in vitro in a standard cellular assay.

24. Nucleobases 84,813 to 84,839 of SEQ ID NO:2
In certain embodiments, nucleobases 84,813-84,839 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 84,813-84,839 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs:542, 2286, 2672, 2748, 2824, 2900, 3130, and 3207 are complementary to nucleobases 84,813 to 84,839 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 84,813-84,839 of SEQ ID NO:2 achieve at least a 69% reduction of ATXN2 RNA in vitro in a standard cellular assay.

25. Nucleobases 85,051 to 85,076 of SEQ ID NO:2
In certain embodiments, nucleobases 85,051-85,076 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 85,051-85,076 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 85,051-85,076 of SEQ ID NO:2 achieve at least a 57% reduction of ATXN2 RNA in vitro in a standard cellular assay.

26. Nucleobases 97,618 to 97,643 of SEQ ID NO:2
In certain embodiments, nucleobases 97,618-97,643 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 97,618-97,643 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 97,618-97,643 of SEQ ID NO:2 achieve at least 72% reduction of ATXN2 RNA in vitro in a standard cellular assay.

27. Nucleobases 119,023 to 119,048 of SEQ ID NO:2
In certain embodiments, nucleobases 119,023-119,048 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 119,023-119,048 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 119,023-119,048 of SEQ ID NO:2 achieve at least a 69% reduction of ATXN2 RNA in vitro in a standard cellular assay.

28. Nucleobases 132,161 to 132,195 of SEQ ID NO:2
In certain embodiments, nucleobases 132,161-132,195 of SEQ ID NO:2 comprise a hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 132,161 to 132,195 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ ID NOs: 1927, 2002, 2381, 2458, 2763, 2839, 2915, and 2991 are complementary to nucleobases 132,161 to 132,195 of SEQ ID NO:2.

In certain embodiments, modified oligonucleotides complementary to nucleobases 132,161-132,195 of SEQ ID NO:2 achieve at least 78% reduction of ATXN2 RNA in vitro in a standard cellular assay.

29. Nucleobases 139,271 to 139,303 of SEQ ID NO:2
In certain embodiments, nucleobases 139,271-139,303 of SEQ ID NO:2 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 139,271-139,303 of SEQ ID NO:2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

The nucleobase sequences of SEQ.

In certain embodiments, modified oligonucleotides complementary to nucleobases 139,271-139,303 of SEQ ID NO:2 achieve at least a 61% reduction of ATXN2 RNA in vitro in a standard cellular assay.

30. Nucleobases 1,075 to 1,146 of SEQ ID NO: 1
In certain embodiments, nucleobases 1,075-1,146 of SEQ ID NO:1 comprise the hotspot region. In certain embodiments, the modified oligonucleotide is complementary to nucleobases 1,075-1,146 of SEQ ID NO:1. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmer is a 5-10-5 MOE gapmer. In certain embodiments, the gapmer is a 6-10-4 MOE gapmer. In certain embodiments, the internucleoside linkages of modified oligonucleotides are phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss. In certain embodiments, phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in 5′ to 3′ order. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in the order 5′ to 3′: soooossssssssssooss.

SEQ. and 3301 are complementary to nucleobases 1,075-1,146 of SEQ ID NO:1.

In certain embodiments, modified oligonucleotides complementary to nucleobases 1,075-1,146 of SEQ ID NO:1 achieve at least a 49% reduction in ATXN2 mRNA in vitro in a standard cellular assay.

NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE Each of the articles and patent publications listed herein is incorporated by reference in its entirety.

While certain compounds, compositions, and methods described herein have been described with specificity according to certain embodiments, the following examples demonstrate the compounds described herein. serves only to explain and is not intended to be limiting. Each of the references listed in this application, GenBank accession numbers, etc., is hereby incorporated by reference in its entirety.

The Sequence Listing attached to this application identifies each sequence as either "RNA" or "DNA" as appropriate, and indeed sequences that may be modified with any combination of chemical modifications. Those skilled in the art will readily appreciate that the designation of modified oligonucleotides as "RNA" or "DNA" to describe them is, in certain instances, arbitrary. For example, oligonucleotides containing 2′-OH sugar moieties and nucleosides containing a thymine base can be used as DNA with modified sugars (2′-OH in place of one 2′-H of DNA) or as modified It can be described as RNA with a base (thymine (methylated uracil) instead of uracil in RNA). Accordingly, the nucleic acid sequences provided herein are natural or modified, including, but not limited to, those nucleic acids having modified nucleobases, including but not limited to those in the sequence listing. It is intended to include nucleic acids, including any combination of RNA and/or DNA. By way of further example, without limitation, oligomeric compounds having the nucleobase sequence "ATCGATCG", whether modified or unmodified, include, but are not limited to, those having the sequence "AUCGAUCG" as well as some and some RNA bases such as "AUCGATCG" and oligomeric compounds with other modified nucleobases such as "AT ^m CGAUCG" , encompasses any oligomeric compound having such a nucleobase sequence, and ^m C denotes a cytosine base containing a methyl group at the 5-position.

Certain compounds described herein (e.g., modified oligonucleotides) possess one or more asymmetric centers and thus as enantiomers, diastereomers, and (R) or (S) Other stereoisomeric configurations occur that can be defined in terms of absolute stereochemistry as α or β such as sugar anomers, or (D) or (L) such as amino acids. Compounds provided herein that are noted or described as having a particular stereoisomeric configuration include only the indicated compounds. Compounds described herein that are noted or described with an undefined stereochemistry include all such stereochemistry, including their stereorandom and optionally pure forms, unless otherwise specified. Includes possible isomers. Likewise, all tautomeric forms of the compounds herein are also included unless otherwise indicated. Unless otherwise indicated, the compounds disclosed herein are intended to include the corresponding salt forms.

またはその塩。
〔項３８〕
以下の式の修飾されたオリゴヌクレオチド、

またはその塩。
〔項３９〕
以下の式の修飾されたオリゴヌクレオチド、

またはその塩。
〔項４０〕
以下の式の修飾されたオリゴヌクレオチド、

またはその塩。
〔項４１〕
以下の式の修飾されたオリゴヌクレオチド、

またはその塩。
〔項４２〕
以下の式の修飾されたオリゴヌクレオチド、

またはその塩。
〔項４３〕
前記式のナトリウム塩である、請求項３７～４２のいずれかに記載の修飾されたオリゴヌクレオチド。
〔項４４〕
以下の式の修飾されたオリゴヌクレオチド。

〔項４５〕
以下の式の修飾されたオリゴヌクレオチド。

〔項４６〕
以下の式の修飾されたオリゴヌクレオチド。

〔項４７〕
以下の式の修飾されたオリゴヌクレオチド。

〔項４８〕
以下の式の修飾されたオリゴヌクレオチド。

〔項４９〕
以下の式の修飾されたオリゴヌクレオチド。

〔項５０〕
請求項３７～４９のいずれかに記載の修飾されたオリゴヌクレオチドのキラル的に濃縮された集団であって、前記集団が、特定の立体化学的配置を有する少なくとも１つの特定のホスホロチオエートヌクレオシド間結合を含む修飾されたオリゴヌクレオチドについて濃縮されている、前記キラル的に濃縮された集団。
〔項５１〕
前記集団が、（Ｓｐ）配置を有する少なくとも１つの特定のホスホロチオエートヌクレオシド間結合を含む修飾されたオリゴヌクレオチドについて濃縮されている、請求項５０に記載のキラル的に濃縮された集団。
〔項５２〕
前記集団が、（Ｒｐ）配置を有する少なくとも１つの特定のホスホロチオエートヌクレオシド間結合を含む修飾されたオリゴヌクレオチドについて濃縮されている、請求項５０または５１に記載のキラル的に濃縮された集団。
〔項５３〕
前記集団が、各ホスホロチオエートヌクレオシド間結合において特定の、独立して選択される立体化学的配置を有する修飾されたオリゴヌクレオチドについて濃縮されている、請求項５０に記載のキラル的に濃縮された集団。
〔項５４〕
前記集団が、各ホスホロチオエートヌクレオシド間結合において前記（Ｓｐ）配置を有する修飾されたオリゴヌクレオチドについて濃縮されている、請求項５３に記載のキラル的に濃縮された集団。
〔項５５〕
前記集団が、各ホスホロチオエートヌクレオシド間結合において前記（Ｒｐ）配置を有する修飾されたオリゴヌクレオチドについて濃縮されている、請求項５３に記載のキラル的に濃縮された集団。
〔項５６〕
前記集団が、５’から３’の方向で、Ｓｐ－Ｓｐ－Ｒｐ配置における少なくとも３個の隣接するホスホロチオエートヌクレオシド間結合を有する修飾されたオリゴヌクレオチドについて濃縮されている、請求項５０または請求項５３に記載のキラル的に濃縮された集団。
〔項５７〕
請求項３７～４９のいずれかに記載の修飾されたオリゴヌクレオチドの集団であって、前記修飾されたオリゴヌクレオチドの前記ホスホロチオエートヌクレオシド間結合の全てが、ステレオランダムである、前記修飾されたオリゴヌクレオチドの集団。
〔項５８〕
請求項３７～４９のいずれかに記載の修飾されたオリゴヌクレオチド及び薬学的に許容される希釈剤または担体を含む薬学的組成物。
〔項５９〕
前記薬学的に許容される希釈剤が、人工脳脊髄液である、請求項３６または５８に記載の薬学的組成物。
〔項６０〕
前記薬学的組成物が、前記修飾されたオリゴヌクレオチド及び人工脳脊髄液から本質的になる、請求項５９に記載の薬学的組成物。
〔項６１〕
請求項３６または５８～６０のいずれかに記載の薬学的組成物を動物に投与することを含む方法。
〔項６２〕
ＡＴＸＮ２に関連する疾患を治療する方法であって、ＡＴＸＮ２に関連する疾患を有するかまたはそれを発症する危険性のある個体に、治療有効量の、請求項３６または５８～６０のいずれかに記載の薬学的組成物を投与し、それによりＡＴＸＮ２に関連する前記疾患を治療することを含む、前記方法。
〔項６３〕
ＡＴＸＮ２に関連する前記疾患が、神経変性疾患である、請求項６２に記載の方法。
〔項６４〕
前記神経変性疾患が、脊髄小脳失調症２型（ＳＣＡ２）、筋萎縮性側索硬化症（ＡＬＳ）、及びパーキンソニズムのうちのいずれかである、請求項６３に記載の方法。
〔項６５〕
前記神経変性疾患の少なくとも１つの症状または特徴が、改善される、請求項６４に記載の方法。
〔項６６〕
前記症状または特徴が、運動失調、神経障害、及び凝集体形成のうちのいずれかである、請求項６５に記載の方法。
〔項６７〕
以下の式の修飾されたオリゴヌクレオチドを含むオリゴマー化合物であって、
Ｇｅｓ Ｔｅｏ Ａｅｏ ｍＣｅｏ Ｔｅｏ Ｔｄｓ Ｔｄｓ Ｔｄｓ ｍＣｄｓ Ｔｄｓ ｍＣｄｓ Ａｄｓ Ｔｄｓ Ｇｄｓ Ｔｄｓ Ｇｅｏ ｍＣｅｏ Ｇｅｓ Ｇｅｓ ｍＣｅ（配列番号１７１４）、式中、
Ａ＝アデニン核酸塩基、
ｍＣ＝５－メチルシトシン核酸塩基、
Ｇ＝グアニン核酸塩基、
Ｔ＝チミン核酸塩基、
ｅ＝２’－ＭＯＥ修飾された糖、
ｄ＝２’－デオキシリボース糖、
ｓ＝ホスホロチオエートヌクレオシド間結合、及び
ｏ＝ホスホジエステルヌクレオシド間結合である、前記オリゴマー化合物。
〔項６８〕
以下の式の修飾されたオリゴヌクレオチドを含むオリゴマー化合物であって、ｍＣｅｓ
Ｔｅｏ Ｇｅｏ ｍＣｅｏ Ｔｄｓ Ａｄｓ Ａｄｓ ｍＣｄｓ Ｔｄｓ Ｇｄｓ Ｇｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ Ｇｅｏ ｍＣｅｏ ｍＣｅｏ ｍＣｅｓ Ｔｅｓ Ｔｅ（配列番号１２５５）、式中、
Ａ＝アデニン核酸塩基、
ｍＣ＝５－メチルシトシン核酸塩基、
Ｇ＝グアニン核酸塩基、
Ｔ＝チミン核酸塩基、
ｅ＝２’－ＭＯＥ修飾された糖、
ｄ＝２’－デオキシリボース糖、
ｓ＝ホスホロチオエートヌクレオシド間結合、及び
ｏ＝ホスホジエステルヌクレオシド間結合である、前記オリゴマー化合物。
〔項６９〕
以下の式の修飾されたオリゴヌクレオチドを含むオリゴマー化合物であって、Ｔｅｓ Ｇｅｏ Ｔｅｏ Ａｅｏ ｍＣｅｏ Ｔｅｏ Ｔｄｓ ｍＣｄｓ Ａｄｓ ｍＣｄｓ Ａｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ Ｇｄｓ Ｇｄｓ Ａｅｏ Ｇｅｓ ｍＣｅｓ ｍＣｅ（配列番号１１８５）、式中、
Ａ＝アデニン核酸塩基、
ｍＣ＝５－メチルシトシン核酸塩基、
Ｇ＝グアニン核酸塩基、
Ｔ＝チミン核酸塩基、
ｅ＝２’－ＭＯＥ修飾された糖、
ｄ＝２’－デオキシリボース糖、
ｓ＝ホスホロチオエートヌクレオシド間結合、及び
ｏ＝ホスホジエステルヌクレオシド間結合である、前記オリゴマー化合物。
〔項７０〕
以下の式の修飾されたオリゴヌクレオチドを含むオリゴマー化合物であって、Ｔｅｓ Ｇｅｏ Ｇｅｏ Ａｅｏ Ｔｅｏ Ｔｅｏ ｍＣｄｓ Ｔｄｓ Ｇｄｓ Ｔｄｓ Ａｄｓ
ｍＣｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ ｍＣｅｏ Ｔｅｓ ｍＣｅｓ Ａｅ（配列番号３２３５）、式中、
Ａ＝アデニン核酸塩基、
ｍＣ＝５－メチルシトシン核酸塩基、
Ｇ＝グアニン核酸塩基、
Ｔ＝チミン核酸塩基、
ｅ＝２’－ＭＯＥ修飾された糖、
ｄ＝２’－デオキシリボース糖、
ｓ＝ホスホロチオエートヌクレオシド間結合、及び
ｏ＝ホスホジエステルヌクレオシド間結合である、前記オリゴマー化合物。
〔項７１〕
以下の式の修飾されたオリゴヌクレオチドを含むオリゴマー化合物であって、ｍＣｅｓ
ｍＣｅｏ Ｔｅｏ Ａｅｏ Ｔｅｏ ｍＣｅｏ Ａｄｓ Ｔｄｓ ｍＣｄｓ Ａｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ ｍＣｄｓ ｍＣｄｓ Ａｅｏ Ｇｅｓ Ｇｅｓ Ｇｅ（配列番号１５８）、式中、
Ａ＝アデニン核酸塩基、
ｍＣ＝５－メチルシトシン核酸塩基、
Ｇ＝グアニン核酸塩基、
Ｔ＝チミン核酸塩基、
ｅ＝２’－ＭＯＥ修飾された糖、
ｄ＝２’－デオキシリボース糖、
ｓ＝ホスホロチオエートヌクレオシド間結合、及び
ｏ＝ホスホジエステルヌクレオシド間結合である、前記オリゴマー化合物。
〔項７２〕
以下の式の修飾されたオリゴヌクレオチドを含むオリゴマー化合物であって、Ｔｅｓ ｍＣｅｏ Ｔｅｏ Ｇｅｏ Ｔｅｓ Ａｄｓ ｍＣｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ Ｔｄｓ ｍＣｄｓ Ｔｄｓ ｍＣｄｓ Ａｄｓ Ｔｅｏ Ｇｅｏ Ｔｅｓ Ｇｅｓ ｍＣｅ（配列番号２５４４）、式中、
Ａ＝アデニン核酸塩基、
ｍＣ＝５－メチルシトシン核酸塩基、
Ｇ＝グアニン核酸塩基、
Ｔ＝チミン核酸塩基、
ｅ＝２’－ＭＯＥ修飾された糖、
ｄ＝２’－デオキシリボース糖、
ｓ＝ホスホロチオエートヌクレオシド間結合、及び
ｏ＝ホスホジエステルヌクレオシド間結合である、前記オリゴマー化合物。
〔項７３〕
前記修飾されたオリゴヌクレオチドが、ＲＮＡｉ化合物である、請求項３に記載のオリゴマー化合物。
〔項７４〕
前記ＲＮＡｉ化合物が、ｓｓＲＮＡまたはｓｉＲＮＡである、請求項７３に記載のオリゴマー化合物。 Compounds described herein include variations in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated element. For example, compounds herein containing hydrogen atoms include all possible deuterium substitutions for each of the ¹ H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but ^are not limited to, ^2H or ^3H for 1H, ^13C or ^14C for ^12C , ^15N for ^14N , ¹⁷ O or ¹⁸ O in place of ¹⁶ O, and ³³ S, ³⁴ S, ³⁵ S, or ³⁶ S in place of ³² S. In certain embodiments, non-radioactive isotopic substitutions can impart novel properties to oligomeric compounds that are beneficial for use as therapeutics or research tools. In certain embodiments, radioisotope substitution can make compounds suitable for research or diagnostic purposes, such as imaging.
Claims as filed
[Item 1]
An oligomeric compound comprising a modified oligonucleotide consisting of 12-50 linked nucleosides, wherein the nucleobase sequence of said modified oligonucleotide is at least 90% complementary to an isometric portion of ATXN2 nucleic acid. , said oligomeric compound, wherein said modified oligonucleotide comprises at least one modification selected from modified sugars and modified internucleoside linkages.
[Section 2]
consisting of 12-50 linked nucleosides and at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 of any of the nucleobase sequences of SEQ ID NOs:30-3319 , a modified oligonucleotide having a nucleobase sequence comprising at least 18, at least 19, or at least 20 contiguous nucleobases.
[Item 3]
consisting of 12-50 linked nucleosides, 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 1. An oligomeric compound comprising a modified oligonucleotide having a nucleobase sequence comprising a portion of 1, at least 18, at least 19, or at least 20 contiguous nucleobases, said portion comprising:
an isometric portion of nucleobases 2,455 to 2,483 of SEQ ID NO: 1;
an isometric portion of nucleobases 4,393 to 4,424 of SEQ ID NO: 1;
an isometric portion of nucleobases 4,413 to 4,437 of SEQ ID NO: 1;
an isometric portion of nucleobases 4,525 to 4,554 of SEQ ID NO:2;
an isometric portion of nucleobases 4,748 to 4,771 of SEQ ID NO:2;
an isometric portion of nucleobases 9,927 to 9,954 of SEQ ID NO:2;
an isometric portion of nucleobases 10,345 to 10,368 of SEQ ID NO:2;
an isometric portion of nucleobases 17,153 to 17,182 of SEQ ID NO:2;
an isometric portion of nucleobases 18,680 to 18,702 of SEQ ID NO:2;
an isometric portion of nucleobases 23,251 to 23,276 of SEQ ID NO:2;
an isometric portion of nucleobases 28,081 to 28,105 of SEQ ID NO:2;
an isometric portion of nucleobases 28,491 to 28,526 of SEQ ID NO:2;
an isometric portion of nucleobases 28,885 to 28,912 of SEQ ID NO:2;
an isometric portion of nucleobases 32,328 to 32,352 of SEQ ID NO:2;
an isometric portion of nucleobases 32,796 to 32,824 of SEQ ID NO:2;
an isometric portion of nucleobases 32,809 to 32,838 of SEQ ID NO:2;
an isometric portion of nucleobases 36,308 to 36,334 of SEQ ID NO:2;
an isometric portion of nucleobases 36,845 to 36,872 of SEQ ID NO:2;
an isometric portion of nucleobases 49,147 to 49,173 of SEQ ID NO:2;
an isometric portion of nucleobases 57,469 to 57,494 of SEQ ID NO:2;
an isometric portion of nucleobases 82,848 to 82,874 of SEQ ID NO:2;
an isometric portion of nucleobases 83,784 to 83,813 of SEQ ID NO:2;
an isometric portion of nucleobases 84,743 to 84,782 of SEQ ID NO:2;
an isometric portion of nucleobases 84,813 to 84,839 of SEQ ID NO:2;
an isometric portion of nucleobases 85,051 to 85,076 of SEQ ID NO:2;
an isometric portion of nucleobases 97,618 to 97,643 of SEQ ID NO:2;
an isometric portion of nucleobases 119,023 to 119,048 of SEQ ID NO:2;
an isometric portion of nucleobases 132,161 to 132,195 of SEQ ID NO:2;
an isometric portion of nucleobases 139,271 to 139,303 of SEQ ID NO:2, or
said oligo which is complementary to an isometric portion of nucleobases 1,075 to 1,146 of SEQ ID NO:1
mer compound.
[Item 4]
said modified oligonucleotide is at least 80%, at least 85%, at least 90% the nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2 when measured over the entire nucleobase sequence of said modified oligonucleotide; An oligomeric compound according to any preceding claim having nucleobase sequences that are at least 95%, or 100% complementary.
[Item 5]
The oligomeric compound of any of claims 1-4, wherein said modified oligonucleotide comprises at least one modified nucleoside.
[Item 6]
6. The oligomeric compound of Claim 5, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a modified sugar moiety.
[Item 7]
7. The oligomeric compound of Claim 6, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety.
[Item 8]
Said modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety having a 2′-4′ bridge, said 2′-4′ bridge being —O—CH ₂ — and — 8. An oligomeric compound according to claim 7, selected from O--CH(CH ₃ )--.
[Item 9]
The oligomeric compound of any of claims 5-8, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety.
[Item 10]
wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety comprising a 2'-MOE modified sugar or a 2'-OMe modified sugar; Item 9. The oligomeric compound according to item 9.
[Item 11]
The oligomeric compound of any of claims 5-10, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising a sugar surrogate.
[Item 12]
12. The oligomeric compound of claim 11, wherein said modified oligonucleotide comprises at least one modified nucleoside comprising sugar surrogates selected from morpholinos and PNAs.
[Item 13]
The modified oligonucleotide is
a 5' region consisting of 1-5 linked 5' region nucleosides;
a central region consisting of 6-10 linked central region nucleosides, and
having a sugar motif comprising a 3' region consisting of 1-5 linked 3' region nucleosides;
Clause 1, wherein each of said 5' region nucleosides and each of said 3' region nucleosides comprises a modified sugar moiety and each of said central region nucleosides comprises an unmodified 2'-deoxyribosyl sugar moiety. 13. The oligomeric compound according to any one of -12.
[Item 14]
The oligomeric compound of any of claims 1-13, wherein said modified oligonucleotide comprises at least one modified internucleoside linkage.
[Item 15]
15. The oligomeric compound of claim 14, wherein each internucleoside linkage of said modified oligonucleotide is a modified internucleoside linkage.
[Item 16]
16. The oligomeric compound of claim 14 or 15, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage.
[Item 17]
17. The oligomeric compound of claim 14 or 16, wherein said modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.
[Item 18]
18. The oligomeric compound of any of claims 14, 16 or 17, wherein each internucleoside linkage is either a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.
[Item 19]
The oligomeric compound of any of claims 1-18, wherein said modified oligonucleotide comprises at least one modified nucleobase.
[Item 20]
20. The oligomeric compound of claim 19, wherein said modified nucleobase is 5-methylcytosine.
[Item 21]
wherein said modified oligonucleotide consists of 12-30, 12-22, 12-20, 14-20, 15-25, 16-20, 18-22, or 18-20 linked nucleosides. Item 21. The oligomeric compound according to any one of items 1 to 20.
[Item 22]
An oligomeric compound according to any preceding claim, wherein said modified oligonucleotide consists of 18 or 20 linked nucleosides.
[Item 23]
An oligomeric compound according to any one of claims 1 to 22, consisting of said modified oligonucleotide.
[Item 24]
An oligomeric compound according to any preceding claim, comprising a conjugate group comprising a conjugate moiety and a conjugate linker.
[Item 25]
25. The oligomeric compound of claim 24, wherein said conjugate group comprises a GalNAc cluster comprising 1-3 GalNAc ligands.
[Item 26]
26. The oligomeric compound of claim 24 or 25, wherein said conjugate linker consists of a single bond.
[Item 27]
26. The oligomeric compound of claim 25, wherein said conjugate linker is cleavable.
[Item 28]
28. The oligomeric compound of claim 27, wherein said conjugate linker comprises 1-3 linker nucleosides.
[Item 29]
The oligomeric compound of any of claims 24-28, wherein said conjugate group is attached to said modified oligonucleotide at the 5' end of said modified oligonucleotide.
[Item 30]
The oligomeric compound of any of claims 24-28, wherein said conjugate group is attached to said modified oligonucleotide at the 3' end of said modified oligonucleotide.
An oligomeric compound according to any of claims 1-30, comprising terminal groups.
[Item 32]
An oligomeric compound according to any preceding claim, wherein said oligomeric compound is a single-stranded oligomeric compound.
[Item 33]
The oligomeric compound of any of claims 1-27 or 29-31, wherein said oligomeric compound is free of linker nucleosides.
[Item 34]
An oligomeric duplex comprising an oligomeric compound according to any one of claims 1-31 or 33.
[Item 35]
An antisense compound comprising or consisting of an oligomeric compound according to any one of claims 1 to 33 or an oligomeric duplex according to claim 34.
[Item 36]
A pharmaceutical composition comprising an oligomeric compound according to any one of claims 1 to 33 or an oligomeric duplex according to claim 34 and a pharmaceutically acceptable carrier or diluent.
[Item 37]
modified oligonucleotides of the formula:

Or its salt.
[Item 38]
modified oligonucleotides of the formula:

Or its salt.
[Item 39]
modified oligonucleotides of the formula:

Or its salt.
[Item 40]
modified oligonucleotides of the formula:

Or its salt.
[Item 41]
modified oligonucleotides of the formula:

Or its salt.
[Item 42]
modified oligonucleotides of the formula:

Or its salt.
[Item 43]
43. The modified oligonucleotide of any of claims 37-42, which is the sodium salt of said formula.
[Item 44]
A modified oligonucleotide of the formula:

[Item 45]
A modified oligonucleotide of the formula:

[Item 46]
A modified oligonucleotide of the formula:

[Item 47]
A modified oligonucleotide of the formula:

[Item 48]
A modified oligonucleotide of the formula:

[Item 49]
A modified oligonucleotide of the formula:

[Item 50]
50. A chirally enriched population of modified oligonucleotides according to any of claims 37-49, wherein said population has at least one specific phosphorothioate internucleoside linkage with a specific stereochemical configuration. Said chirally enriched population enriched for modified oligonucleotides comprising:
[Item 51]
51. The chirally enriched population of claim 50, wherein said population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) configuration.
[Item 52]
52. The chirally enriched population of claim 50 or 51, wherein said population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Rp) configuration.
[Item 53]
51. The chirally enriched population of claim 50, wherein said population is enriched for modified oligonucleotides having specific, independently selected stereochemical configurations at each phosphorothioate internucleoside linkage.
[Item 54]
54. The chirally enriched population of claim 53, wherein said population is enriched for modified oligonucleotides having said (Sp) configuration at each phosphorothioate internucleoside linkage.
[Item 55]
54. The chirally enriched population of claim 53, wherein said population is enriched for modified oligonucleotides having said (Rp) configuration at each phosphorothioate internucleoside linkage.
[Item 56]
Claim 50 or claim 53, wherein said population is enriched for modified oligonucleotides having at least three adjacent phosphorothioate internucleoside linkages in the Sp-Sp-Rp configuration in the 5' to 3' direction. A chirally enriched population as described in .
[Item 57]
50. The population of modified oligonucleotides of any of claims 37-49, wherein all of said phosphorothioate internucleoside linkages of said modified oligonucleotides are stereorandom. group.
[Item 58]
A pharmaceutical composition comprising the modified oligonucleotide of any of claims 37-49 and a pharmaceutically acceptable diluent or carrier.
[Item 59]
59. The pharmaceutical composition of claim 36 or 58, wherein said pharmaceutically acceptable diluent is artificial cerebrospinal fluid.
[Item 60]
60. The pharmaceutical composition of claim 59, wherein said pharmaceutical composition consists essentially of said modified oligonucleotide and artificial cerebrospinal fluid.
[Item 61]
61. A method comprising administering the pharmaceutical composition of any of claims 36 or 58-60 to an animal.
[Item 62]
61. A method of treating a disease associated with ATXN2, wherein a therapeutically effective amount of any of claims 36 or 58-60 is administered to an individual having or at risk of developing a disease associated with ATXN2. and thereby treating said disease associated with ATXN2.
[Item 63]
63. The method of claim 62, wherein said disease associated with ATXN2 is a neurodegenerative disease.
[Item 64]
64. The method of claim 63, wherein the neurodegenerative disease is any of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and parkinsonism.
[Item 65]
65. The method of claim 64, wherein at least one symptom or feature of said neurodegenerative disease is ameliorated.
[Item 66]
66. The method of claim 65, wherein the symptom or feature is any of ataxia, neuropathy, and aggregate formation.
[Item 67]
An oligomeric compound comprising a modified oligonucleotide of the formula:
Ges Teo Aeo mCeo Teo Tds Tds Tds mCds Tds mCds Ads Tds Gds Tds Geo mCeo Ges Ges mCe (SEQ ID NO: 1714), wherein
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
Said oligomeric compound, wherein o = a phosphodiester internucleoside linkage.
[Item 68]
An oligomeric compound comprising a modified oligonucleotide of the formula mCes
Teo Geo mCeo Tds Ads Ads mCds Tds Gds Gds Tds Tds Tds Tds Geo mCeo mCeo mCes Te (SEQ ID NO: 1255), wherein
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
Said oligomeric compound, wherein o = a phosphodiester internucleoside linkage.
[Item 69]
An oligomeric compound comprising a modified oligonucleotide of the formula Tes Geo Teo Aeo mCeo Teo Tds mCds Ads mCds Ads Tds Tds Tds Gds Gds Aeo Ges mCes mCe (SEQ ID NO: 1185), wherein
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
Said oligomeric compound, wherein o = a phosphodiester internucleoside linkage.
[Item 70]
An oligomeric compound comprising a modified oligonucleotide of the formula Tes Geo Geo Aeo Teo Teo mCds Tds Gds Tds Ads
mCds Tds Tds Tds Tds mCeo Tes mCes Ae (SEQ ID NO: 3235), wherein
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
Said oligomeric compound, wherein o = a phosphodiester internucleoside linkage.
[Item 71]
An oligomeric compound comprising a modified oligonucleotide of the formula mCes
mCeo Teo Aeo Teo mCeo Ads Tds mCds Ads Tds Tds Tds Tds Tds mCds mCds Aeo Ges Ges Ge (SEQ ID NO: 158), wherein
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
Said oligomeric compound, wherein o = a phosphodiester internucleoside linkage.
[Item 72]
An oligomeric compound comprising a modified oligonucleotide of the formula Tes mCeo Teo Geo Tes Ads mCds Tds Tds Tds Tds Tds mCds Tds mCds Ads Teo Geo Tes Ges mCe (SEQ ID NO: 2544), wherein
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
Said oligomeric compound, wherein o = a phosphodiester internucleoside linkage.
[Item 73]
4. The oligomeric compound of claim 3, wherein said modified oligonucleotide is an RNAi compound.
[Item 74]
74. The oligomeric compound of claim 73, wherein said RNAi compound is ssRNA or siRNA.

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Further, where specific embodiments are provided, the inventors have contemplated the general application of those specific embodiments. For example, disclosure of an oligonucleotide with a particular motif provides reasonable support for additional oligonucleotides with the same or similar motifs. Further, for example, if a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position would be preferred unless otherwise indicated.

Example 1: Effect of 5-10-5 MOE Gapmer with Mixed Internucleoside Linkage to Human ATXN2 RNA in Vitro, Single Dose Modified oligonucleotides complementary to human ATXN2 nucleic acid were designed to -04 cells were tested for their effect on ATXN2 RNA. SCA2-04 is a patient fibroblast cell line with 34 CAG repeats. Modified oligonucleotides were tested in a series of experiments with similar culture conditions.

SCA2-04 cells cultured at a density of 20,000 cells per well were treated with modified oligos at concentrations of 2,000 nM or 7,000 nM, as indicated in the table below, relative to untreated controls. Transfected using electroporation with or without modified oligonucleotides. Approximately 24 hours later, RNA was isolated from the cells and ATXN2 RNA levels were measured by quantitative real-time PCR. human primer probe set hAtaxin_LTS01321 (forward sequence ATATGGACTCCAGTTATGCAAAAAAGA designated herein as SEQ ID NO: 10; reverse sequence TCGCCATTTCACTTTAGCACTGA designated herein as SEQ ID NO: 11; probe sequence ATGCTTTTACTGACTCTGC ) was used to measure RNA levels. ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent ATXN2 RNA levels relative to untreated control cells.

Modified oligonucleotides marked with an asterisk (*) target the amplicon region of the primer probe set. Additional assays may be used to measure potency and efficacy of oligonucleotides targeting amplicon regions.

The modified oligonucleotides in the table below are 5-10-5 MOE gapmers. Gapmers are 20 nucleobases long, with a central gap segment containing 10 2′-deoxynucleosides and wing segments at both the 5′ and 3′ ends containing 5 2′-MOE nucleosides. Adjacent. The gapmer sugar motif is (5′ to 3′)eeeeeddddddddddeeeee, where “d” represents the 2′-deoxyribose sugar and “e” represents the 2′-MOE modified sugar. Internucleoside linkages are mixed phosphodiester and phosphorothioate internucleoside linkages. The gapmer internucleoside linkage motif is (5' to 3') soooossssssssssooss, where 'o' represents a phosphodiester internucleoside linkage and 's' represents a phosphorothioate internucleoside linkage. Each cytosine residue is 5-methylcytosine. "Initiation site" indicates the 5'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence.

Each modified oligonucleotide listed in the table below is complementary to the human ATXN2 nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, as indicated. "N/A" indicates that the modified oligonucleotide is not complementary to that particular nucleic acid with 100% complementarity. As shown below, modified oligonucleotides complementary to the nucleobase sequence of human ATXN2 reduced the amount of human ATXN2 RNA.

Example 2 Effect of 5-10-5 MOE Gapmers with Mixed Internucleoside Linkages on Human ATXN2 RNA Expression in Vitro, Single Dose Modified oligonucleotides complementary to human ATXN2 nucleic acids were designed and They were tested for their effect on ATXN2 RNA in vitro.

A431 cells cultured at a density of 10,000 cells per well were transfected via free uptake with or without modified oligonucleotides at a concentration of 5,000 nM relative to untreated controls. effected. Approximately 24 hours later, RNA was isolated from the cells and ATXN2 RNA levels were measured by quantitative real-time PCR. Human primer probe set RTS5049 (forward sequence CTACAGTCCTCAGCAGTTCC designated herein as SEQ ID NO: 13, reverse sequence GCCATCATTCTAGCATTACCCT designated herein as SEQ ID NO: 14, probe sequence ATCAGCCCCCTTGTTCAGCATGTG designated herein as SEQ ID NO: 15 ) was used to measure RNA levels. ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent control ATXN2 RNA levels relative to untreated control cells.

The modified oligonucleotides in the table below are 5-10-5 MOE gapmers. Gapmers are 20 nucleobases long, with a central gap segment containing 10 2′-deoxynucleosides and wing segments at both the 5′ and 3′ ends containing 5 2′-MOE nucleosides. Adjacent. The gapmer sugar motif is (5' to 3')eeeeeddddddddddeeeee, where 'd' represents the 2'-deoxyribose sugar and 'e' represents the 2'-MOE modified sugar. Internucleoside linkages are mixed phosphodiester and phosphorothioate linkages. The gapmer internucleoside linkage motif is (5' to 3') soooossssssssssooss, where 'o' represents a phosphodiester internucleoside linkage and 's' represents a phosphorothioate internucleoside linkage. Each cytosine residue is 5-methylcytosine. "Start site" indicates the 5'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence.

Each modified oligonucleotide listed in the table below is complementary to the human ATXN2 nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, as indicated. "N/A" indicates that the modified oligonucleotide is not complementary to that particular nucleic acid with 100% complementarity. As shown below, modified oligonucleotides complementary to the nucleobase sequence of human ATXN2 RNA reduced the amount of human ATXN2 RNA.

Example 3 Effect of 5-10-5 MOE Gapmers with Mixed Internucleoside Linkages on Human ATXN2 RNA Expression in Vitro, Single Dose Modified oligonucleotides complementary to human ATXN2 nucleic acids were designed and They were tested for their effect on ATXN2 RNA in vitro.

A431 cells cultured at a density of 10,000 cells per well were electroporated with or without modified oligonucleotides at a concentration of 6,000 nM versus untreated controls. transfected. Approximately 24 hours later, RNA was isolated from the cells and ATXN2 RNA levels were measured by quantitative real-time PCR as described in Example 2. Results are presented in the table below as percent ATXN2 RNA levels relative to untreated control cells. Modified oligonucleotides marked with an asterisk (*) target the amplicon region of the primer probe set. Additional assays may be used to measure potency and efficacy of oligonucleotides targeting amplicon regions.

The modified oligonucleotides in the table below are 5-10-5 MOE gapmers. Gapmers are 20 nucleobases long, with a central gap segment containing 10 2′-deoxynucleosides and wing segments at both the 5′ and 3′ ends containing 5 2′-MOE nucleosides. Adjacent. The gapmer sugar motif is (5' to 3')eeeeeddddddddddeeeee, where 'd' represents the 2'-deoxyribose sugar and 'e' represents the 2'-MOE modified sugar. Internucleoside linkages are mixed phosphodiester and phosphorothioate linkages. The gapmer internucleoside linkage motif is (5' to 3') soooossssssssssooss, where 'o' represents a phosphodiester internucleoside linkage and 's' represents a phosphorothioate internucleoside linkage. Each cytosine residue is 5-methylcytosine. "Start site" indicates the 5'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence.

Each modified oligonucleotide listed in the table below is complementary to the human ATXN2 nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, as indicated. "N/A" indicates that the modified oligonucleotide is not complementary to that particular nucleic acid with 100% complementarity. As shown below, modified oligonucleotides complementary to the nucleobase sequence of human ATXN2 reduced the amount of human ATXN2 RNA.

Example 4: Effect of 5-10-5 MOE gapmers with mixed internucleoside linkages on human ATXN2 RNA expression at multiple doses in vitro. Various doses were tested in -04 cells. Cells were seeded at a density of 20,000 cells per well and modified Oligonucleotides were transfected using electroporation. After a treatment period of approximately 24 hours, total RNA was isolated from the cells and ATXN2
RNA levels were measured by quantitative real-time PCR. RNA levels were measured using the human ATXN2 primer probe set hAtaxin_LTS01321 (described in Example 1 above). ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent ATXN2 RNA expression relative to untreated control cells. As shown in the table below, ATXN2 RNA levels were reduced in a dose-dependent manner in modified oligonucleotide-treated cells. _IC50s were calculated using the log(inhibitor) vs. response-variable slope (4 parameters) formula using Prism6 software.

Example 5: Effect of 5-10-5 MOE gapmers with mixed internucleoside linkages on human ATXN2 RNA expression at multiple doses in vitro. Various doses in cells were tested. Cells were seeded at a density of 10,000 cells per well and modified oligonucleotides at concentrations of 0.44 μM, 1.33 μM, 4.00 μM, and 12.00 μM, as specified in the table below. were transfected by free uptake at . After a treatment period of approximately 24 hours, total RNA was isolated from cells and ATXN2 RNA levels were measured by quantitative real-time PCR. RNA levels were measured using the human ATXN2 primer probe set RTS5049 (described in Example 2 above). ATXN2 according to total RNA content as measured by RIBOGREEN®
RNA levels were regulated. Results are presented in the table below as percent ATXN2 RNA expression relative to untreated controls. As shown in the table below, ATXN2 RNA levels were reduced in a dose-dependent manner in modified oligonucleotide-treated cells. The _IC50 was calculated as log(inhibitor) versus response-variable slope (4 parameters)' formula u sing
Calculated using Prism6 software.

Example 6 In Vitro, Effect of 5-10-5 MOE Gapmers with Mixed Internucleoside Linkages to Human ATXN2 at Multiple Dosing A variety of doses were tested. Cells were seeded at a density of 10,000 cells per well and modified oligonucleotides at concentrations of 0.094, 0.375, 1.500, and 6,000 μM, as specified in the table below. were transfected by free uptake at . After a treatment period of approximately 24 hours, total RNA was isolated from cells and ATXN2 RNA levels were measured by quantitative real-time PCR. RNA levels were measured using the human ATXN2 primer probe set RTS5049 (described in Example 2 above). ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent ATXN2 RNA expression relative to untreated control cells. As shown in the table below, ATXN2 RNA levels were reduced in a dose-dependent manner in modified oligonucleotide-treated cells. _IC50s were calculated using the log(inhibitor) vs. response-variable slope (4 parameters) formula using Prism6 software.

Example 7: Design of gapmers with mixed internucleoside linkages complementary to human ATXN2 RNA Modified oligonucleotides complementary to human ATXN2 nucleic acids were designed. The modified oligonucleotides in the table below are gapmers. Gapmers have a central gap segment containing 2'-deoxynucleosides and are flanked by wing segments at both the 5' and 3' ends containing 2'-MOE nucleosides. Internucleoside linkages are mixed phosphodiester internucleoside linkages and phosphorothioate internucleoside linkages. Each cytosine residue is 5-methylcytosine. The sequence and chemical notation columns specify sequences including 5-methylcytosine, sugar chemistry, and internucleoside linkage chemistry, subscript 'd' representing a 2'-deoxyribose sugar, subscript ' The subscript 'o' represents a phosphodiester internucleoside linkage, the subscript 's' represents a phosphorothioate internucleoside linkage, and the subscript 's' represents a 2'-MOE modified sugar; The preceding 'm' subscript indicates 5-methylcytosine. "Initiation site" indicates the 5'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence.

Each modified oligonucleotide listed in the table below is complementary to the human ATXN2 nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, as indicated. "N/A" indicates that the modified oligonucleotide is not complementary to that particular nucleic acid with 100% complementarity.

Example 8 Effect of MOE Gapmers with Mixed Internucleoside Linkages to Human ATXN2 in Vitro, Multiple Dosing tested. Cells were seeded at a density of 11,000 cells per well and modified at concentrations of 0.023, 0.094, 0.375, 1.500, or 6,000 μM as specified in the table below. were transfected by free uptake with oligonucleotides. After a treatment period of approximately 48 hours, total RNA was isolated from cells and ATXN2 RNA levels were measured by quantitative real-time PCR. Human ATXN2 primer probe set RTS5051 (forward sequence TCCA designated herein as SEQ ID NO: 16
RNA levels were measured using GTAGCAAGGACCAGT, reverse sequence CAATACTGTTCTGTCTGGGAGA designated herein as SEQ ID NO: 17, probe sequence ACTGACCACTGATGACCACGTTCC designated herein as SEQ ID NO: 18). ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent ATXN2 RNA expression relative to untreated control cells. As shown in the table below, ATXN2 RNA levels were reduced in a dose-dependent manner in modified oligonucleotide-treated cells. _IC50s were calculated using the log(inhibitor) vs. response-variable slope (4 parameters) formula using Prism6 software.

Example 9 Effect of Modified Oligonucleotides on Human ATXN2 at Multiple Dosing In Vitro Selected modified oligonucleotides from the above examples were tested at various doses in SH-SY5Y cells. Cells were seeded at a density of 35,000 cells per well and modified at concentrations of 0.023, 0.094, 0.375, 1.500, or 6,000 μM as specified in the table below. were transfected by free uptake with oligonucleotides. After a treatment period of approximately 24 hours, total RNA was isolated from cells and ATXN2 RNA levels were measured by quantitative real-time PCR. RNA levels were measured using the human ATXN2 primer probe set RTS5051 (described in Example 8 herein). ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent ATXN2 RNA relative to untreated controls. As shown in the table below, ATXN2 RNA levels were reduced in a dose-dependent manner in modified oligonucleotide-treated cells. _IC50s were calculated using the log(inhibitor) vs. response-variable slope (4 parameters) formula using Prism6 software.

Example 10 Effect of Modified Oligonucleotides on Rhesus ATXN2 RNA Expression in Vitro, Multiple Dosing Various doses were tested in monkey cells. Cells were seeded at a density of 20,000 cells per well and modified at concentrations of 0.023, 0.094, 0.375, 1.500, or 6,000 μM as specified in the table below. were transfected by free uptake with oligonucleotides. After a treatment period of approximately 24 hours, total RNA was isolated from cells and ATXN2 RNA levels were measured by quantitative real-time PCR. RNA levels were measured using the human ATXN2 primer probe set RTS5051 (described in Example 8 herein). ATXN2 RNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in the table below as percent ATXN2 RNA expression relative to untreated control cells. As shown in the table below, ATXN2 RNA levels were reduced in a dose-dependent manner in modified oligonucleotide-treated cells. _IC50s were calculated using the log(inhibitor) vs. response-variable slope (4 parameters) formula using Prism6 software.

Example 11: Design of 5-8-5 MOE gapmers with mixed internucleoside linkages complementary to human ATXN2 RNA Modified oligonucleotides complementary to human ATXN2 nucleic acid were designed. The modified oligonucleotides in the table below are 5-8-5 MOE gapmers. Gapmers are 18 nucleobases long, with a central gap segment containing 8 2′-deoxynucleosides and wing segments at both the 5′ and 3′ ends containing 5 2′-MOE nucleosides. Adjacent. The gapmer sugar motif is (5′ to 3′)eeeeeddddddddeeeee, where “d” represents the 2′-deoxyribose sugar and “e” represents the 2′-MOE modified sugar. Internucleoside linkages are mixed phosphodiester and phosphorothioate linkages. The gapmer internucleoside linkage motif is (5' to 3') sooosssssssssooss, where 'o' represents a phosphodiester internucleoside linkage and 's' represents a phosphorothioate internucleoside linkage. Each cytosine residue is 5-methylcytosine. "Initiation site" indicates the 5'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence.

Each modified oligonucleotide listed in the table below is complementary to the human ATXN2 nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, as indicated. "N/A" indicates that the modified oligonucleotide is not complementary to that particular nucleic acid with 100% complementarity.

Example 12: Design of 4-10-6 MOE gapmers with mixed internucleoside linkages complementary to human ATXN2 RNA Modified oligonucleotides complementary to human ATXN2 nucleic acid were designed. The modified oligonucleotides in the table below are 4-10-6 MOE gapmers. Gapmers are 20 nucleobases long, with a central gap segment containing 8 2′-deoxynucleosides and wing segments at both the 5′ and 3′ ends containing 5 2′-MOE nucleosides. Adjacent. The gapmer sugar motif is (5′ to 3′)eeeeddddddddddeeeeee, where “d” represents the 2′-deoxyribose sugar and “e” represents the 2′-MOE modified sugar. Internucleoside linkages are mixed phosphodiester and phosphorothioate linkages. The gapmer internucleoside linkage motif is (5' to 3') sooossssssssssoooss, where 'o' represents a phosphodiester internucleoside linkage and 's' represents a phosphorothioate internucleoside linkage. Each cytosine residue is 5-methylcytosine. "Initiation site" indicates the 5'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is complementary in the human nucleic acid sequence.

Each modified oligonucleotide listed in the table below is complementary to the human ATXN2 nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, as indicated. "N/A" indicates that the modified oligonucleotide is not complementary to that particular nucleic acid with 100% complementarity.

Example 13 Tolerability of Modified Oligonucleotides Complementary to Human ATXN2 RNA in Wild-Type Mice, 3 Hour FOB Assessment was assessed for tolerability. The comparator oligonucleotides 564122, 564127, 564133, 564143, 564150, 564188, 564210, 564216 described herein above and in WO2015/143246 were also tested. Wild-type C57/B16 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.
Groups of 3 mice received PBS as a negative control for each experiment (specified in separate tables below). Three hours after injection, mice were evaluated according to 7 different criteria. Criteria were: (1) mice were bright, alert and responsive; (2) mice were standing or hunched without stimulation; (3) mice were performing any movement without stimulation. (4) mice exhibited forward movement after being lifted; (5) mice exhibited arbitrary movements after being lifted; (7) Normal breathing. For each of the 7 criteria, mice were given a subscore of 0 if the criterion was met and 1 if not (Functional Observational Global Assessment Score or FOB). After assessing all seven criteria, scores were summed for each mouse and averaged within each treatment group. Results are presented in the table below as mean scores for each treatment group.

Example 14 Tolerability of Modified Oligonucleotides Complementary to Human ATXN2 RNA in Wild-Type Rats, FOB Evaluation The modified oligonucleotides described above were tested in Sprague Dawley rats to demonstrate oligonucleotide tolerance. evaluated. Sprague Dawley rats each received a single intrathecal (IT) dose of 3 mg of the oligonucleotides listed in the table below. Each treatment group consisted of 4 rats. Groups of 4 rats received PBS as a negative control for each experiment (specified in separate tables below). At 3 hours post-injection, each rat was assessed for movement in 7 different parts of the body. The seven body parts are: (1) rat tail, (2) rat rearward posture, (3) rat hind legs, (4) rat hind legs, (5) rat front legs, (6) rat front legs. Posture, (7) the head of the rat. For each of the 7 different body parts, each rat was given a subscore of 0 if the body part was moving or 1 if the body part was not moving (Functional Observation Global Assessment Score or FOB ). After assessing all seven criteria, scores were summed for each rat and averaged within each treatment group. The results are presented in the table below.

Example 15 Activity of Modified Oligonucleotides Complementary to Human ATXN2 RNA in Transgenic Mice In a BAC-ATXN2-Q22 transgenic mouse model generated by using RP11-798L5, 16 kb of 5′ flanking genomic sequence, and 3 kb of 3′ flanking genomic sequence that contained the entire 150 kb human ATXN2 locus with repeats. tested (Dansithong et al., PLoS Genetics, 2015).

hATXN2 mice were divided into groups of 3-4 mice each. Two groups were tested with each compound. hATXN2 mice each received a single intracerebroventricular (ICV) administration of 350 μg of modified oligonucleotide. A PBS-injected group, which served as a control group, was compared to the oligonucleotide-treated group. Two weeks later, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time PCR analysis of human ATXN2 RNA expression measurement using the primer probe set hAtaxin_LTS01321 described in Example 1 above. did. Results are expressed as percent ATXN2 RNA expression relative to PBS control, normalized to GADPH. As shown in the table below, treatment with modified oligonucleotides resulted in significance of ATXN2 RNA compared to PBS controls.

Claims (20)

A modified oligonucleotide of the following chemical structure:

Or its salt. 2. The modified oligonucleotide of claim 1 , which is a sodium or potassium salt . The following chemical structures:

modified oligonucleotides. The formula below:
Tes Geo Geo Aeo Teo Teo mCds Tds Gds Tds Ads mCds Tds Tds Tds Tds mCeo Tes mCes Ae
(SEQ ID NO: 3235)
An oligomeric compound comprising a modified oligonucleotide of
A = adenine nucleobase,
mC = 5-methylcytosine nucleobase,
G = guanine nucleobase,
T = thymine nucleobase,
e = 2'-MOE modified sugar,
d = 2'-deoxyribose sugar,
s = phosphorothioate internucleoside linkage, and
o = phosphodiester internucleoside linkage
The oligomeric compound which is The population of modified oligonucleotides of any of claims 1-3 or the population of oligomeric compounds of claim 4 , wherein all of said phosphorothioate internucleoside linkages of said modified oligonucleotides are stereo said population of modified oligonucleotides being random. A modified oligonucleotide according to any one of claims 1 to 3 , an oligomeric compound according to claim 4, a population of modified oligonucleotides according to claim 5, or an oligomeric compound according to claim 5. A pharmaceutical composition comprising a population and a pharmaceutically acceptable diluent . 7. The pharmaceutical composition of claim 6 , wherein said pharmaceutically acceptable diluent is artificial cerebrospinal fluid or phosphate buffered saline (PBS) . 8. The pharmaceutical composition of claim 7 , wherein said pharmaceutical composition consists essentially of said modified oligonucleotide or said oligomeric compound and artificial cerebrospinal fluid. 8. The pharmaceutical composition of claim 7, wherein said pharmaceutical composition consists essentially of said modified oligonucleotide or said oligomeric compound and PBS. 8. The pharmaceutical composition of claim 7, wherein said pharmaceutical composition consists essentially of said population of modified oligonucleotides or said population of oligomeric compounds and artificial cerebrospinal fluid. 8. The pharmaceutical composition of claim 7, wherein said pharmaceutical composition consists essentially of said population of modified oligonucleotides or said population of oligomeric compounds and PBS. A pharmaceutical composition for treatment in an animal, comprising: a modified oligonucleotide according to any one of claims 1 to 3, an oligomeric compound according to claim 4, a modified oligonucleotide according to claim 5 A population of oligonucleotides, a population of oligomeric compounds according to claim 5, or a pharmaceutical composition according to any of claims 6-11 . 13. The pharmaceutical composition of claim 12, wherein said animal is human. 13. The pharmaceutical composition of claim 12, wherein said treatment comprises reducing expression of ATXN2 in cells. 15. The pharmaceutical composition of claim 14, wherein said cells are human cells. A modified oligonucleotide according to any one of claims 1 to 3, an oligomeric compound according to claim 4, a population of modified oligonucleotides according to claim 5, for treating a disease associated with ATXN2. , a pharmaceutical composition comprising a population of oligomeric compounds according to claim 5, or a pharmaceutical composition according to any one of claims 6-11.
A pharmaceutical composition comprising
A therapeutically effective amount of said modified oligonucleotide, oligomeric compound, population of modified oligonucleotides, population of oligomeric compounds, or pharmaceutical Said pharmaceutical composition , wherein administering said composition treats said disease associated with ATXN2. 17. The pharmaceutical composition of claim 16 , wherein said disease associated with ATXN2 is a neurodegenerative disease. 18. The pharmaceutical composition of claim 17, wherein the neurodegenerative disease is any of spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and parkinsonism. 19. The pharmaceutical composition of claim 17 or claim 18, wherein at least one symptom or feature of said neurodegenerative disease is ameliorated. 20. The pharmaceutical composition of claim 19, wherein said symptom or feature is any of ataxia, neuropathy, and aggregate formation.