Methods for the synthesis of linkage-modified oligomeric compounds - Patents.com

Abstract

The present disclosure provides a method for synthesizing modified oligonucleotides having at least one modified internucleoside linking group, and oligomeric compounds comprising modified oligonucleotides, including oligomeric compounds that are antisense agents or portions thereof.In certain embodiments, the present disclosure provides a stabilized formulation of a certain sulfonyl azide for use in synthesizing oligonucleotides that comprise one or more sulfonyl phosphoramidate bonds.Some embodiments provide a stabilized composition of high-energy reagents that can be used in the synthesis of modified oligonucleotides, thereby enabling their safe process-scale preparation.

Description

[Technical field]

[0001] Sequence Listing This application has been filed in electronic format together with a sequence listing, which is provided in a file entitled DVCM0048WOSEQ_ST25.txt, created on June 29, 2022, and having a size of 1 kb. The information in the electronic format of this sequence listing is incorporated herein by reference in its entirety.

[0002] The present disclosure provides stabilized reagent compositions for the synthesis of oligomeric compounds, including modified oligonucleotides having at least one modified internucleoside linkage group, including oligomeric compounds that are antisense agents or portions thereof. [Background technology]

[0003] The principle behind antisense technology is that antisense compounds hybridize to target nucleic acid and regulate the amount, activity, and / or function of target nucleic acid. For example, in certain cases, antisense compounds cause changes in the transcription or translation of the target. Such regulation of expression can be achieved, for example, by inhibition based on degradation or occupancy of target RNA. One example of regulation of RNA target function by degradation is RNase H-based degradation of target RNA upon hybridization with DNA-like antisense compounds.

[0004] Another example of regulating gene expression by target degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing via a mechanism that utilizes the RNA-induced silencing complex (RISC). A further example of regulating RNA target function is by occupancy-based mechanisms, such as the mechanism naturally used by microRNAs. MicroRNAs are small non-coding RNAs that control the expression of protein-coding RNAs. Binding of an antisense compound to a microRNA prevents the microRNA from binding to its messenger RNA target, thus interfering with the function of the microRNA. MicroRNA mimics can enhance native microRNA function. Certain antisense compounds alter the splicing of pre-mRNAs. Another example of regulating gene expression is the use of antisense compounds in CRISPR systems. Regardless of the specific mechanism, sequence specificity makes antisense compounds attractive as tools for target validation and gene functioning, as well as therapeutic agents to selectively regulate the expression of genes involved in disease pathogenesis.

[0005] Antisense technology is an effective means for regulating the expression of one or more specific gene products, and therefore may prove to be uniquely useful in some therapeutic, diagnostic, and research applications.Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, tolerance, pharmacokinetics, or affinity of target nucleic acid.Conjugate groups may be attached to antisense compounds to enhance one or more properties, such as pharmacokinetics, pharmacodynamics, and uptake into cells and / or tissues of interest.

[0006] Oligomeric compounds, including oligonucleotides, are chemically synthesized in a multi-step process. Substituted sulfonyl azides are useful reagents for the synthesis of bond-modified oligonucleotides, but they can be high-energy materials and can be dangerous to handle, especially at manufacturing scale. The development of new stabilized reagent compositions and reaction conditions to improve the potentially dangerous chemistry on scale remains a significant challenge. Summary of the Invention

[0007] The disclosure provides methods for synthesizing oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) that comprise modified oligonucleotides comprised of linked nucleosides linked via internucleoside linking groups, at least one of which has the formula I: [ka] wherein X and R are as defined herein. The method may include adding a stabilizing material to the composition of the energetic reagent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] 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 of the embodiments as claimed. As used herein, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting.

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

[0010] It is understood that the sequence defined by each SEQ ID NO contained herein is independent of any modification to the sugar moiety, internucleoside linkage, or nucleobase. Thus, the compound defined by a SEQ ID NO may independently include one or more modifications to the sugar moiety, internucleoside linkage, or nucleobase. Although the sequence listing attached to this application identifies each sequence as either "RNA" or "DNA" as appropriate, in practice, the sequences may be modified with any combination of chemical modifications. Those skilled in the art will readily appreciate that designations such as "RNA" or "DNA" to describe modified oligonucleotides are arbitrary in certain instances. For example, an oligonucleotide containing a nucleoside containing a 2'-OH (H) sugar moiety and a thymine base may be described as a DNA with a modified sugar (2'-OH instead of one 2'-H of DNA) or as an RNA with a modified base (thymine (methylated uracil) instead of uracil of RNA). Thus, the nucleic acid sequences provided herein, including but not limited to those in the Sequence Listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids with modified nucleobases. By way of further example and without limitation, a modified oligonucleotide having the nucleobase sequence "ATCGATCG" encompasses any modified oligonucleotide having such a nucleobase sequence, whether modified or unmodified, including such compounds containing RNA bases, e.g., compounds having the sequence "AUCGAUCG," as well as compounds having any DNA base and any RNA base, e.g., "AUCGATCG," as well as other modified nucleobases, e.g., "AT m CGAUCG” (in the formula, m Examples of modified oligonucleotides include, but are not limited to, modified oligonucleotides having a cytosine base containing a methyl group at the 5-position.

[0011] As used herein, "2'-substituted" with reference to a furanosyl sugar moiety or a nucleoside that includes a furanosyl sugar moiety means that the furanosyl sugar moiety or a nucleoside that includes a furanosyl sugar moiety includes a substituent other than H or OH at the 2' position and is a non-bicyclic furanosyl sugar moiety. A 2'-substituted furanosyl sugar moiety, when in the context of an oligonucleotide, does not include additional substituents at other positions on the furanosyl sugar moiety, other than the nucleobase and / or internucleoside linkage(s).

[0012] As used herein, "4' substituted" with reference to a furanosyl sugar moiety or a nucleoside that includes a furanosyl sugar moiety means that the furanosyl sugar moiety or a nucleoside that includes a furanosyl sugar moiety includes a substituent other than H at the 4' position and is a non-bicyclic furanosyl sugar moiety. A 4' substituted furanosyl sugar moiety, when in the context of an oligonucleotide, does not include additional substituents at other positions on the furanosyl sugar moiety, other than the nucleobase and / or internucleoside linkage(s).

[0013] As used herein, "5' substituted" with reference to a furanosyl sugar moiety or a nucleoside that includes a furanosyl sugar moiety means that the furanosyl sugar moiety or a nucleoside that includes a furanosyl sugar moiety includes a substituent other than H at the 5' position and is a non-bicyclic furanosyl sugar moiety. A 5' substituted furanosyl sugar moiety, when in the context of an oligonucleotide, does not include additional substituents at other positions on the furanosyl sugar moiety, other than the nucleobase and / or internucleoside linkage(s).

[0014] As used herein, "administration" or "administering" refers to a route of introducing a compound or composition provided herein into a subject to perform its intended function. Examples of administration routes that may be used include, but are not limited to, administration by inhalation, subcutaneous injection, intrathecal injection, and oral administration.

[0015] As used herein, "antisense activity" refers to any detectable and / or measurable change attributable to the hybridization of an antisense oligonucleotide to its target nucleic acid. In certain embodiments, antisense activity is a decrease 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 level or target protein level in the absence of the antisense oligonucleotide.

[0016] As used herein, "antisense agent" means an antisense oligonucleotide or an oligonucleotide duplex that includes an antisense oligonucleotide.

[0017] As used herein, "antisense compound" means an antisense oligonucleotide or an oligonucleotide duplex that contains an antisense oligonucleotide.

[0018] As used herein, "antisense oligonucleotide" refers to an oligonucleotide that is complementary to a target nucleic acid and can achieve at least one antisense activity.Antisense oligonucleotides include, but are not limited to, RNAi antisense modified oligonucleotides and RNase H antisense modified oligonucleotides.In certain embodiments, antisense oligonucleotides are paired with sense oligonucleotides to form oligonucleotide duplexes.In certain embodiments, antisense oligonucleotides are unpaired and are single-stranded antisense oligonucleotides.In certain embodiments, antisense oligonucleotides include conjugate groups.

[0019] As used herein, an "artificial mRNA compound" is a modified oligonucleotide having a nucleobase sequence containing one or more codons, or a portion thereof.

[0020] As used herein, "bicyclic nucleoside" or "BNA" refers to a nucleoside that comprises a bicyclic sugar moiety. As used herein, "bicyclic sugar" or "bicyclic sugar moiety" refers to a modified sugar moiety that comprises two rings, where the second ring is formed via a bridge that connects two of the atoms in the first ring, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety, and the bicyclic sugar moiety is a modified bicyclic furanosyl sugar moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

[0021] As used herein, "capping reagent" refers to a reagent that is effective for protecting hydroxyl groups during synthesis of oligonucleotides, for example, synthesis on a solid support. In certain embodiments, the capping reagent may be acetic anhydride. The capping reagent may be delivered in a composition that includes a base and a solvent. For example, the capping reagent composition may include acetic anhydride and acetonitrile, or pyridine, N-methylimidazole (NMI), and acetonitrile.

[0022] As used herein, "cEt" or "constrained ethyl" or "cEt sugar moiety" means a bicyclic sugar moiety, where the first ring of the bicyclic sugar moiety is a ribosyl sugar moiety and the second ring of the bicyclic sugar is formed through a bridge connecting the 4' and 2' carbons, the bridge having the formula 4'-CH(CH3)-O-2', and the methyl group of the bridge is in the S configuration. The cEt bicyclic sugar moiety is in the β-D configuration.

[0023] As used herein, "complementary" with reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and another nucleic acid or one or more regions thereof are capable of hydrogen bonding with each other when the nucleobase sequences of the oligonucleotide and the other nucleic acid are aligned in opposite orientations. Complementary nucleobases are nucleobase pairs that can form hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methylcytosine (C) and 5-methylcytosine (D) and 5-methylcytosine (E). m Examples of complementary bases include amino acids 1 (C) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at every nucleoside. Rather, some mismatches are permitted. As used herein, "fully complementary" or "100% complementary" with reference to an oligonucleotide means that such oligonucleotide is complementary to another oligonucleotide or nucleic acid at every nucleoside of the oligonucleotide.

[0024] As used herein, "conjugate group" refers to the grouping of atoms consisting of a conjugate moiety and a conjugate linker.

[0025] As used herein, "conjugate moiety" means a grouping of atoms that modifies one or more properties of a molecule, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance, compared to the same molecule lacking the conjugate moiety.

[0026] As used herein, "conjugate linker" means a group of atoms that includes at least one bond.

[0027] As used herein, "CRISPR compound" refers to a modified oligonucleotide that includes a DNA recognition portion and a tracrRNA recognition portion. As used herein, a "DNA recognition portion" is a nucleobase sequence that is complementary to a DNA target. As used herein, a "tracrRNA recognition portion" is a nucleobase sequence that is bound to or can bind to a tracrRNA. The tracrRNA recognition portion of a crRNA can bind to the tracrRNA via hybridization or covalent binding.

[0028] As used herein, "cytotoxic" or "cytotoxicity" in the context of the effects of an oligomeric compound or parent oligomeric compound on cultured cells means at least a two-fold increase in caspase activation following application of 10 μM or less of an oligomeric compound or parent oligomeric compound to cultured cells, as compared to cells cultured under the same conditions but not applied with the oligomeric compound or parent oligomeric compound. In certain embodiments, cytotoxicity is measured using a standard in vitro cytotoxicity assay.

[0029] As used herein, a "deoxy region" refers to a region of 5-12 contiguous nucleotides in which at least 70% of the nucleosides are stereo-standard DNA nucleosides. In certain embodiments, each nucleoside is selected from a stereo-standard DNA nucleoside (a nucleoside containing a β-D-2'-deoxyribosyl sugar moiety), a stereo-non-standard nucleoside of Formulas I-VII, a bicyclic nucleoside, and a substituted stereostandard nucleoside. In certain embodiments, the deoxy region supports RNase H activity. In certain embodiments, the deoxy region is the gap of a gapmer.

[0030] As used herein, a "double-stranded antisense compound" means an antisense compound that comprises two oligomeric compounds that are complementary to each other and form a duplex, wherein one of the two oligomeric compounds comprises an antisense oligonucleotide.

[0031] As used herein, "expression" includes all functions by which a gene's coded information is converted into structures present and operative in a cell. Such structures include, but are not limited to, transcription and translation products. As used herein, "modulation of expression" refers to any change in the amount or activity of a transcription or translation product of a gene. Such a change can be any amount of increase or decrease compared to the expression level prior to modulation.

[0032] As used herein, a "gapmer" refers to an oligonucleotide having a central region containing multiple nucleosides that support RNase H cleavage, located between the 5' and 3' regions. As used herein, the nucleosides of the 5' and 3' regions each contain a 2'-substituted furanosyl sugar moiety or a bicyclic sugar moiety, and the 3'- and 5'-most nucleosides of the central region each contain a sugar moiety independently selected from a 2'-deoxyfuranosyl sugar moiety or a sugar surrogate. The position of the central region refers to the order of the nucleosides in the central region, counted starting from the 5' end of the central region. Thus, the 5'-most nucleoside of the central region is at position 1 of the central region. The "central region" may be referred to as the "gap," and the "5' region" and "3' region" may be referred to as the "wings." The gap of a gapmer is a deoxy region.

[0033] As used herein, "hepatotoxicity" in the context of mice means plasma ALT levels that are greater than 300 units / liter. Hepatotoxicity of an oligomeric compound or parent oligomeric compound administered to a mouse is determined by measuring plasma ALT levels in the mouse 24 hours to 2 weeks after at least one dose of the compound at 1 to 150 mg / kg.

[0034] As used herein, "hepatotoxicity" in the human context means plasma ALT levels that are greater than 150 units / liter. Hepatotoxicity of an oligomeric compound or parent oligomeric compound administered to a human is determined by measuring plasma ALT levels in the human 24 hours to 2 weeks after at least one dose of 10 to 300 mg of the compound.

[0035] As used herein, "hybridization" refers to the pairing or annealing of complementary oligonucleotides and / or nucleic acids. Although not limited to a particular mechanism, the most common hybridization mechanism involves hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementary nucleobases.

[0036] As used herein, "inhibiting expression or activity" refers to a reduction or blocking of expression or activity compared to expression or activity in an untreated or control sample, and does not necessarily indicate a complete elimination of expression or activity.

[0037] As used herein, "internucleoside bond" or "internucleoside linking group" refers to a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. As used herein, "modified internucleoside bond" refers to any internucleoside bond other than a naturally occurring phosphodiester internucleoside bond. "Phosphorothioate bond" refers to a modified internucleoside bond in which one of the non-bridging oxygen atoms of the phosphodiester is replaced with a sulfur atom. The modified internucleoside bond may or may not contain a phosphorus atom. "Neutral internucleoside bond" refers to a modified internucleoside bond that does not have a negatively charged phosphate in an aqueous buffer solution at pH=7.0. The modified internucleoside bond may optionally contain a conjugate group.

[0038] As used herein, "linked nucleosides" are nucleosides that are joined in contiguous sequence (ie, there are no additional nucleosides between the linked nucleosides).

[0039] As used herein, "maximum tolerated dose" refers to the highest dose of compound that does not cause unacceptable side effects.In certain embodiments, maximum tolerated dose is the highest dose of modified oligonucleotide that does not cause ALT elevation of 3 times the upper normal limit as measured by standard assay.

[0040] As used herein, "modulating" refers to changing or adjusting a characteristic in a cell, tissue, organ, or organism.

[0041] As used herein, "MOE" means O-methoxyethyl. "2'-MOE" or "2'-O-methoxyethyl" means a 2'-OCH2CH2OCH3 group at the 2'-position of a furanosyl ring. In certain embodiments, the 2'-OCH2CH2OCH3 group replaces the 2'-OH group of a ribosyl ring or replaces the 2'-H in a 2'-deoxyribosyl ring. A "2'-MOE sugar moiety" is a sugar moiety having a 2'-OCH2CH2OCH3 group in place of the 2'-OH group of a furanosyl sugar moiety. Unless otherwise indicated, the 2'-MOE sugar moiety is in the β-D ribosyl configuration.

[0042] As used herein, a "2'-OMe sugar moiety" is a sugar moiety having a 2'-CH group in place of the 2'-OH group of a furanosyl sugar moiety. Unless otherwise indicated, the 2'-OMe sugar moiety is in the β-D ribosyl configuration and is a "stereostandard 2'OMe sugar moiety."

[0043] As used herein, a "2'-F sugar moiety" is a sugar moiety having a 2'-F group in place of the 2'-OH group of a furanosyl sugar moiety. Unless otherwise indicated, the 2'-F sugar moiety is in the β-D ribosyl configuration and is a "stereostandard 2'-F sugar moiety."

[0044] As used herein, "motif" means a pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages in an oligonucleotide.

[0045] As used herein, "naturally occurring" means found in nature.

[0046] As used herein, "nucleobase" refers to 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 atomic group that can pair with at least one unmodified nucleobase. A universal base is a nucleobase that can pair with any one of these five unmodified nucleobases. 5-methylcytosine ( m C) is an example of a modified nucleobase.

[0047] As used herein, "nucleobase sequence" means the order of contiguous nucleobases in a nucleic acid or oligonucleotide, independent of any modification of the sugar moieties or internucleoside linkages.

[0048] As used herein, "nucleoside" refers to a moiety that includes a nucleobase and a sugar moiety. The nucleobase and sugar moieties are each independently unmodified or modified. As used herein, "modified nucleoside" refers to a nucleoside that includes a modified nucleobase and / or a modified sugar moiety. The modified nucleoside may include a conjugate group.

[0049] As used herein, "oligomeric compound" means a compound consisting of (1) an oligonucleotide (single-stranded oligomeric compound) or two oligonucleotides hybridized to one another (double-stranded oligomeric compound), and (2) optionally one or more additional features, such as a conjugate group or a terminal group, which may be attached to one or both of the oligonucleotides of the single-stranded oligomeric compound or the double-stranded oligomeric compound.

[0050] As used herein, "oligonucleotide" refers to a chain of linked nucleosides joined via internucleoside linkages, where each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 12 to 80 linked nucleosides, and optionally a conjugate group or a terminal group. As used herein, "modified oligonucleotide" refers to an oligonucleotide in which at least one nucleoside (modified nucleoside) or internucleoside linkage (modified internucleoside linkage) is modified. As used herein, "unmodified oligonucleotide" refers to an oligonucleotide that does not contain any nucleoside or internucleoside modification. An oligonucleotide is an oligomeric compound, and an oligonucleotide may be incorporated into an oligomeric compound with additional features. An oligonucleotide or modified oligonucleotide may include a linker group that links it to a solid support. The linker may be as described in Ravikumar et al., Org. Process Res. Dev. 2008, 12, 3, 399-410.

[0051] As used herein, "oligonucleotide intermediate" refers to a compound or a portion thereof that occurs during the synthesis of an oligonucleotide and will ultimately form a part of such an oligonucleotide. Oligonucleotide intermediates include, but are not limited to, linked nucleosides, internucleoside linkages, conjugate groups, and modifications as described herein, as well as their precursors. In certain embodiments, the oligonucleotide intermediate is a hydroxy group attached to a solid support. In certain embodiments, the oligonucleotide intermediate is several linked nucleosides attached to a solid support.

[0052] As used herein, a "pharmaceutical composition" refers to a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include an antisense compound and an aqueous solution.

[0053] As used herein, "RNAi agent" refers to an antisense agent that acts at least in part through RISC or Ago2 to regulate a target nucleic acid and / or a protein encoded by the target nucleic acid. RNAi agents include, but are not limited to, double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics. RNAi agents may include conjugate groups and / or end groups. In certain embodiments, RNAi agents regulate the amount, activity, and / or splicing of a target nucleic acid. The term RNAi agent excludes antisense agents that act through RNase H.

[0054] As used herein, "RNAi oligonucleotide" means an RNAi antisense modified oligonucleotide or an RNAi sense modified oligonucleotide.

[0055] As used herein, "RNAi antisense modified oligonucleotide" means an oligonucleotide that is complementary to a target sequence and contains a region that contains at least one chemical modification suitable for RNAi.

[0056] As used herein, "RNAi antisense oligomeric compound" means a single-stranded oligomeric compound that is complementary to a target sequence and contains a region that contains at least one chemical modification suitable for RNAi.

[0057] As used herein, "RNAi sense modified oligonucleotide" means an oligonucleotide that contains a region complementary to a region of an RNAi antisense modified oligonucleotide and is capable of forming a duplex with such an RNAi antisense modified oligonucleotide.

[0058] As used herein, "RNAi sense oligomeric compound" means a single-stranded oligomeric compound that contains a region complementary to a region of an RNAi antisense modified oligonucleotide and / or an RNAi antisense oligomeric compound and is capable of forming a duplex with such RNAi antisense modified oligonucleotide and / or RNAi antisense oligomeric compound.

[0059] The duplexes formed by an RNAi sense modified oligonucleotide and / or an RNAi sense oligomeric compound and an RNAi antisense modified oligonucleotide and / or an RNAi antisense oligomeric compound are referred to as double-stranded RNAi compounds (dsRNAi) or short interfering RNA (siRNA).

[0060] As used herein, "RNase H agent" refers to an antisense agent that acts at least in part through RNase H to modulate a target nucleic acid and / or a protein encoded by a target nucleic acid. In certain embodiments, the RNase H agent is single stranded. In certain embodiments, the RNase H agent is double stranded. The RNase H compound may include a conjugate group and / or a terminal group. In certain embodiments, the RNase H agent modulates the amount or activity of a target nucleic acid. The term RNase H agent excludes antisense agents that act primarily through RISC / Ago2.

[0061] As used herein, "RNase H antisense modified oligonucleotide" means an oligonucleotide that is complementary to a target sequence and contains a region that contains at least one chemical modification suitable for RNase H-mediated nucleic acid degradation.

[0062] As used herein, "RNAi compound" refers to an antisense compound that acts at least in part through RISC or Ago2 to regulate a target nucleic acid and / or a protein encoded by the target nucleic acid. RNAi compounds include, but are not limited to, double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics. In certain embodiments, RNAi compounds regulate the amount, activity, and / or splicing of a target nucleic acid. The term RNAi compound excludes antisense oligonucleotides that act through RNase H.

[0063] As used herein, the term "single-stranded" in reference to antisense compounds refers to such compounds that consist of one oligomeric compound, without pairing with a second oligomeric compound to form a duplex.The term "self-complementary" in reference to oligonucleotides refers to an oligonucleotide that at least partially hybridizes with itself.A compound that consists of one oligomeric compound, where the oligonucleotide of the oligomeric compound is self-complementary, is a single-stranded compound.Single-stranded antisense or oligomeric compounds can be bound to complementary oligomeric compounds to form a duplex, and then the compound is no longer single-stranded.

[0064] As used herein, "stabilizing phosphate group" refers to a 5' chemical moiety that provides stabilization of the 5' phosphate portion of the 5' terminal nucleoside of an oligonucleotide under biological conditions relative to the stability of the unmodified 5' phosphate of the unmodified nucleoside. Such stabilization of the 5' phosphate group includes, but is not limited to, resistance to removal by phosphatases. Stabilizing phosphate groups include, but are not limited to, 5'-vinyl phosphonate and 5'-cyclopropyl phosphonate.

[0065] As used herein, "stabilizer" refers to a substance that reduces the risk of explosion when present in a solution, including but not limited to a reaction mixture.

[0066] As used herein, "standard oxidizing agent" refers to an oxidizing agent well understood in the art of oligonucleotide synthesis for oxidizing phosphorus internucleoside linkages, including, but not limited to, a basic solvent, a mixture of a basic solvent such as 3-picoline, pyridine, 2,6-lutidine, and the like with iodine and water, and a mixture of iodine, NMI, a basic solvent, and water. Further examples and descriptions of oxidation methods are described in WO2020236618A1, the disclosure of which is incorporated herein in its entirety.

[0067] As used herein, "standard sulfurizing agent" refers to a reagent well understood in the art of oligonucleotide synthesis for sulfurizing phosphorus internucleoside bond, including but not limited to phenacetyldilsulfide or xanthan hydride. Further examples and explanations of oxidation methods are described in WO2020236618A1, the disclosure of which is incorporated herein in its entirety.

[0068] As used herein, a "stereostandard nucleoside" refers to a nucleoside containing a non-bicyclic furanosyl sugar moiety having the naturally occurring DNA and RNA configuration as shown below. A "stereostandard DNA nucleoside" is a nucleoside containing a β-D-2'-deoxyribosyl sugar moiety. A "stereostandard RNA nucleoside" is a nucleoside containing a β-D-ribosyl sugar moiety. A "substituted stereostandard nucleoside" is a stereostandard nucleoside other than a stereostandard DNA or stereostandard RNA nucleoside. In certain embodiments, R1 is a 2' substituent and R2-R5 are each H. In certain embodiments, the 2' substituent is selected from OMe, F, OCH2CHOCH3, O-alkyl, SMe, or NMA. In certain embodiments, R1-R4 are H and R5 is a 5' substituent selected from methyl, allyl, or ethyl. In certain embodiments, the heterocyclic base moiety Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine. In certain embodiments, the heterocyclic base moiety Bx is other than uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine. [ka]

[0069] As used herein, "stereononstandard nucleosides" refers to nucleosides that include a non-bicyclic furanosyl sugar moiety having a configuration other than that of a stereostandard sugar moiety. In certain embodiments, a "stereononstandard nucleoside" includes a 2'-β-L-deoxyribosyl sugar moiety, a 2'-α-D-deoxyribosyl sugar moiety, a 2'-α-L-deoxyribosyl sugar moiety, a 2'-β-D-deoxyxylosyl sugar moiety, a 2'-β-L-deoxyxylosyl sugar moiety, a 2'-α-D-deoxyxylosyl sugar moiety, a 2'-α-L-deoxyxylosyl sugar moiety, a 2'-fluoro-β-D-arabinosyl sugar moiety, a 2'-fluoro-β-D-xylosyl sugar moiety. 2'-fluoro-α-D-ribosyl sugar moiety, 2'-fluoro-α-D-arabinosyl sugar moiety, 2'-fluoro-α-D-xylosyl sugar moiety, 2'-fluoro-α-L-ribosyl sugar moiety, 2'-fluoro-β-L-xylosyl sugar moiety, 2'-fluoro-α-L-arabinosyl sugar moiety, 2'-fluoro-α-L-xylosyl sugar moiety, 2'-fluoro-β-L-ribosyl sugar moiety, 2'-fluoro-β-L-arabinosyl sugar moiety, 2'-fluoro-β-D-lyxosyl sugar moiety lyxosyl sugar moiety, 2'-fluoro-α-D-lyxosyl sugar moiety, 2'-fluoro-α-L-lyxosyl sugar moiety, 2'-fluoro-β-L-lyxosyl sugar moiety, 2'-O-methyl-β-D-arabinosyl sugar moiety, 2'-O-methyl-β-D-xylosyl sugar moiety, 2'-O-methyl-α-D-ribosyl sugar moiety, 2'-O-methyl-α-D-arabinosyl sugar moiety, 2'-O-methyl-α-D-xylosyl sugar moiety, 2'-O-methyl-α-L-ribosyl sugar moiety a 2'-O-methyl-β-L-xylosyl sugar moiety, a 2'-O-methyl-α-L-arabinosyl sugar moiety, a 2'-O-methyl-α-L-xylosyl sugar moiety, a 2'-O-methyl-β-L-ribosyl sugar moiety, a 2'-O-methyl-β-L-arabinosyl sugar moiety, a 2'-O-methyl-β-D-lyxosyl sugar moiety, a 2'-O-methyl-α-D-lyxosyl sugar moiety, a 2'-O-methyl-α-L-lyxosyl sugar moiety, or a 2'-O-methyl-β-L-lyxosyl sugar moiety.

[0070] As used herein, "stereostandard sugar moiety" means the sugar moiety of a stereostandard nucleoside.

[0071] As used herein, "stereostandard sugar moiety" means the sugar moiety of a stereostandard nucleoside.

[0072] As used herein, "substituted stereononstandard nucleosides" refers to stereononstandard nucleosides that contain substituents other than those corresponding to natural RNA or DNA. In certain embodiments, the substituted stereononstandard nucleoside is a 2'-fluoro-β-D-arabinosyl sugar moiety, a 2'-fluoro-β-D-xylosyl sugar moiety, a 2'-fluoro-α-D-ribosyl sugar moiety, a 2'-fluoro-α-D-arabinosyl sugar moiety, a 2'-fluoro-α-D-xylosyl sugar moiety, a 2'-fluoro-α-L-ribosyl sugar moiety, a 2'-fluoro-β-L-xylosyl sugar moiety, a 2'-fluoro-α-L-ribosyl sugar moiety, a 2'-fluoro-β-L-xylosyl sugar moiety, a 2'-fluoro-α-D-arabinosyl sugar moiety, a 2'-fluoro-α-D-xylosyl sugar moiety, a 2'-fluoro-α-L-ribosyl sugar moiety, a 2'-fluoro-β-L-xyl ...D-xylosyl sugar 2'-fluoro-α-L-arabinosyl sugar moiety, 2'-fluoro-α-L-xylosyl sugar moiety, 2'-fluoro-β-L-ribosyl sugar moiety, 2'-fluoro-β-L-arabinosyl sugar moiety, 2'-fluoro-β-D-lyxosyl sugar moiety, 2'-fluoro-α-D-lyxosyl sugar moiety, 2'-fluoro-α-L-lyxosyl sugar moiety, 2'-fluoro-β-L-lyxosyl Sugar moiety, 2'-O-methyl-β-D-arabinosyl sugar moiety, 2'-O-methyl-β-D-xylosyl sugar moiety, 2'-O-methyl-α-D-ribosyl sugar moiety, 2'-O-methyl-α-D-arabinosyl sugar moiety, 2'-O-methyl-α-D-xylosyl sugar moiety, 2'-O-methyl-α-L-ribosyl sugar moiety, 2'-O-methyl-β-L-xylosyl sugar moiety, 2'-O-methyl-α-L -arabinosyl sugar moiety, 2'-O-methyl-α-L-xylosyl sugar moiety, 2'-O-methyl-β-L-ribosyl sugar moiety, 2'-O-methyl-β-L-arabinosyl sugar moiety, 2'-O-methyl-β-D-lyxosyl sugar moiety, 2'-O-methyl-α-D-lyxosyl sugar moiety, 2'-O-methyl-α-L-lyxosyl sugar moiety, or 2'-O-methyl-β-L-lyxosyl sugar moiety.

[0073] As used herein, "sulfonyl oxidizing agent" refers to an agent capable of effecting the conversion of a phosphite triester to a phosphoramidate. In certain embodiments, the sulfonyl oxidizing agent has the structure [ka] where R is as defined for formula I. In certain embodiments, R is methyl and the sulfonyl oxidizing agent is methanesulfonyl azide ("MsN3").

[0074] As used herein, "sugar moiety" refers to an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" refers to a β-D-ribosyl moiety as found in naturally occurring RNA, or a β-D-2'-deoxyribosyl sugar moiety as found in naturally occurring DNA. As used herein, "modified sugar moiety" or "modified sugar" refers to a sugar surrogate or furanosyl sugar moiety other than β-D-ribosyl or β-D-2'-deoxyribosyl. Modified furanosyl sugar moieties may or may not be modified or substituted at a particular position(s) on the sugar moiety, and they may or may not be stereo-nonstandard sugar moieties. Modified furanosyl sugar moieties include bicyclic and non-bicyclic sugars. As used herein, "sugar surrogate" means a modified sugar moiety that does not contain a furanosyl or tetrahydrofuranyl ring (not a "furanosyl sugar moiety") and that can link a nucleobase to another group, such as an internucleoside linkage, a conjugate group, or a terminal group, in an oligonucleotide. Modified nucleosides containing sugar surrogates can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary oligomeric compounds or nucleic acids.

[0075] As used herein, "target nucleic acid", "target RNA", "target RNA transcript" and "nucleic acid target" refer to the nucleic acid that an oligomeric compound, such as an antisense compound, is designed to affect. In certain embodiments, an oligomeric compound comprises an oligonucleotide having a nucleobase sequence that is complementary to more than one RNA, and only one of these RNAs is the target RNA of the oligomeric compound. In certain embodiments, the target RNA is an RNA present in the species to which the oligomeric compound is administered.

[0076] As used herein, "therapeutic index" refers to the amount of a compound that causes a therapeutic effect compared to the amount that causes toxicity. A compound with a high therapeutic index has strong efficacy and low toxicity. In certain embodiments, increasing the therapeutic index of a compound increases the amount of the compound that can be safely administered.

[0077] Certain embodiments The present disclosure provides the following specific embodiments.

[0078] Embodiment 1: A method for producing a phosphite triester internucleoside linkage comprising contacting a first oligonucleotide intermediate having a phosphite triester internucleoside linkage with an oxidation solution comprising at least one stabilizer and a sulfonyl oxidizing agent to form a second oligonucleotide intermediate having an internucleoside linkage group of formula XIV; [ka] During the ceremony, R is an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C1-C6 alkoxy, a C1-C 20 Alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C 20 The method of claim 1, wherein the alkyl group is selected from alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkynyl, and a conjugate group.

[0079] Embodiment 2: The method of embodiment 1, wherein the sulfonyl oxidizing agent is methanesulfonyl azide (MsN3).

[0080] Embodiment 3: The method of any one of embodiments 1-2, wherein the oxidizing solution comprises the at least one stabilizer.

[0081] Embodiment 4: The method of any one of embodiments 1-2, wherein the oxidizing solution does not contain the at least one stabilizer.

[0082] Embodiment 5: The method of any one of embodiments 1 to 4, wherein the oxidizing solution comprises a solvent selected from acetonitrile, toluene, dichloromethane, pyridine, N-methyl-2-pyrrolidone, and combinations thereof.

[0083] Embodiment 6: The method of any one of embodiments 1 to 5, wherein the at least one stabilizer is selected from naphthalene, sulfolane, and triphenyl phosphate.

[0084] Embodiment 7: The method of any one of embodiments 1 to 5, wherein the at least one stabilizer is triphenyl phosphate (TPP).

[0085] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the at least one stabilizer is TPP.

[0086] Embodiment 9:

[0087] Embodiment 10: The method of any one of embodiments 1-9, wherein at least one stabilizer is a non-crosslinked polymer.

[0088] Embodiment 11: The method of embodiment 10, wherein the non-crosslinked polymer is polystyrene.

[0089]

[0023] Embodiment 12: The residue obtained by evaporating the solvent from the oxidizing solution and the at least one stabilizer has a viscosity of 500 J.g -112. The method of any one of the preceding claims, having a combustion energy of less than 1000 ppm.

[0090]

[0023] Embodiment 13: The residue obtained by evaporating the solvent from the oxidizing solution and the at least one stabilizer has a viscosity of 300 J.g -1 12. The method of any one of the preceding claims, having a combustion energy of less than 1000 ppm.

[0091] Embodiment 14: A method for synthesizing a modified oligonucleotide comprising at least one internucleoside linkage of formula I, comprising: [ka] wherein independently for each internucleoside linkage of Formula I: X is selected from O or S; R is an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C1-C6 alkoxy, a C1-C 20 Alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C 20 selected from alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkynyl, and a conjugate group; The method further comprising: a) providing a solid support having a blocked hydroxyl group attached thereto; b) adding a deblocking agent to the reaction to deblock the blocked hydroxyl groups to provide free hydroxyl groups; c) adding a nucleoside to the reaction for coupling at the free hydroxyl group to provide a phosphite triester linked nucleoside, the nucleoside comprising a phosphoramidite group and a blocked hydroxyl group; d) adding to the reactants 1. Standard oxidizing agents for generating phosphate triester internucleosode linkages; 2. A standard sulfurizing agent to generate thiophosphate triester internucleoside linkages, or 3. a sulfonyl oxidizing agent for generating a sulfonyl phosphoramidate internucleoside linkage, and at least one stabilizer selected from TPP; and e) optionally treating the sufonyl phosphoramidate linkage, the phosphate triester linkage, or the thiophosphate triester linkage with a mixture of capping reagents to cap any unreacted free hydroxyl groups; f) repeating steps b) to e) iteratively a predetermined number of times to provide said modified oligonucleotide, with the proviso that at least one iteration includes step (d)3; g) treating the modified oligonucleotide with triethylamine in acetonitrile; Including, Thereby, the modified oligonucleotide containing at least one internucleoside linkage of formula I is synthesized. The method.

[0092] Embodiment 15: The method of embodiment 14, wherein the sulfonyl oxidizing agent is in an oxidizing solution comprising methanesulfonyl azide and the at least one stabilizer.

[0093] Embodiment 16:

[0094] Embodiment 17: The method of any one of embodiments 15-16, wherein the at least one stabilizer is TPP.

[0095] Embodiment 18: The residue obtained by evaporation of the solvent from the solution comprising the sulfonyl oxidizing agent and the at least one stabilizer has a molecular weight of 500 J.g -1 18. The method of any one of embodiments 14 to 17, having a combustion energy of less than 1000 ppm.

[0096] Embodiment 19: The residue obtained by evaporation of the solvent from the solution comprising the sulfonyl oxidizing agent and the at least one stabilizer has a molecular weight of 300 J.g -1 18. The method of any one of embodiments 14 to 17, having a combustion energy of less than 1000 ppm.

[0097] Embodiment 20: The method of any one of embodiments 14-19, wherein X is O and R is methyl.

[0098] Embodiment 21: The method of any one of embodiments 1 to 20, wherein the modified oligonucleotide comprises 12 to 25 linked nucleosides.

[0099] Embodiment 22: The method of any of embodiments 14 to 21, comprising treating the modified oligonucleotide with ammonium hydroxide to remove protecting groups and cleave the modified oligonucleotide from the solid support.

[0100] Certain compounds In certain embodiments, the compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) that comprise or consist of an oligonucleotide consisting of linked nucleosides and having at least one internucleoside linking group of formula I, [ka] wherein X is selected from O or S; R is an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C1-C6 alkoxy, a C1-C 20 Alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C 20 In certain embodiments, X is O, R is methyl, and the internucleoside linking group of formula I is an internucleoside linking group of formula II below.

[0101] In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide consisting of linked nucleosides, including at least one internucleoside linking group of formula II: [ka] (mesyl phosphoramidate internucleoside linkage), including oligomeric compounds that are antisense agents or portions thereof.

[0102] In certain embodiments, the compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) that comprise or consist of oligonucleotides comprised of linked nucleosides and have at least one internucleoside linking group of formula III. [ka]

[0103] In certain embodiments, the compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) that comprise or consist of an oligonucleotide consisting of linked nucleosides and have at least one internucleoside linking group of formula IV. [ka]

[0104] A modified oligonucleotide comprises at least one modification compared to an unmodified oligonucleotide (i.e., comprises at least one modified nucleoside (including a modified sugar moiety, a stereononstandard nucleoside, and / or a modified nucleobase) and / or at least one modified internucleoside linkage). In certain embodiments, a modified internucleoside linkage is a modified internucleoside linkage group having any of Formulas I-IV. In certain embodiments, the compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) having at least one modified internucleoside linkage group having any of Formulas I-IV.

[0105] A specific process for the synthesis of oligonucleotides The present disclosure provides synthetic methods for preparing oligonucleotides containing at least one modified internucleoside linkage of formula I. [ka]

[0106] The present disclosure also provides a synthetic method for preparing an oligomeric compound comprising such an oligonucleotide, wherein such an oligomeric compound comprises a conjugate moiety attached to the oligonucleotide via a cleavable linker. In certain embodiments, the cleavable linker is a phosphodiester bond. In certain embodiments, an oligonucleotide having both at least one internucleoside bond of formula I and at least one phosphorothioate diester bond and / or at least one phosphodiester bond has one or more desired properties. In certain embodiments, an oligonucleotide having at least one internucleoside bond of formula I is a gapmer. In certain embodiments, an oligonucleotide having at least one internucleoside bond of formula I is used to regulate splicing of a nucleic acid target. In certain embodiments, an oligonucleotide having at least one internucleoside bond of formula I is an RNAi compound. Such an RNAi compound can be double-stranded or single-stranded. Such an oligonucleotide can include any of the features, modified nucleosides, and nucleoside motifs described herein.

[0107] Thus, such oligonucleotides may include any of the modified sugar moieties and / or any of the modified nucleobases described herein. In certain embodiments, the synthetic process described herein is used to synthesize an oligomeric compound comprising a conjugate group. In certain embodiments, the synthetic process described herein is used to synthesize an oligomeric compound comprising a conjugate group comprising one or more N-acetylgalactosamine residues. In certain embodiments, the oligomeric compound synthesized using the process described herein is a gapmer. In certain embodiments, the oligomeric compound synthesized using the process described herein is an RNAi compound. In certain embodiments, the oligomeric compound synthesized using the process described herein is single-stranded. In certain embodiments, the oligomeric compound synthesized using the process described herein is double-stranded. In certain embodiments, the compound synthesized using the process described herein is formulated for administration to an animal.

[0108] Certain Reagents for the Synthesis of Oligonucleotides Containing Sulfonyl Phosphoramidate Internucleoside Linkages. The present disclosure provides certain sulfonyl oxidizing agents, such as sulfonyl azides, for use in the synthesis of oligonucleotides that contain one or more sulfonyl phosphoramidate bonds. The present disclosure further provides stabilizers that can be introduced into the sulfonyl phosphoramidate bond formation reaction. In certain embodiments, the stabilizer can be introduced into the bond formation reaction before, simultaneously with, or subsequent to the introduction of the sulfonyl oxidizing agent, such as sulfonyl azide. In certain embodiments, more than one stabilizer can be introduced into the bond formation reaction. In certain embodiments, the stabilizer can improve the energy events associated with the use of sulfonyl azide. In certain embodiments, the stabilizer can be a sterically bulky compound. In certain embodiments, the stabilizer has low flammability properties. In certain embodiments, the stabilizer has low volatility and can be solid or semi-solid at room temperature. In certain embodiments, the sulfonyl azide and stabilizer can be dissolved in a solvent or solvent mixture before being introduced into the bond formation reaction. Solvents that may be useful according to the present disclosure include, but are not limited to, acetonitrile (MeCN), dichloromethane (DCM), toluene, pyridine, N-methyl-2-pyrrolidone (NMP), and mixtures thereof. Solvents that may be useful according to the present disclosure include, but are not limited to, acetonitrile (MeCN), toluene, dichloromethane (DCM), toluene, pyridine, N-methyl-2-pyrrolidone (NMP), and mixtures thereof. In certain embodiments, the solvent is acetonitrile and toluene. In certain embodiments, the stabilizer may be a solid or liquid at room temperature. Stabilizers that may be useful in the present disclosure include, but are not limited to, naphthalene, sulfolane, and triphenyl phosphate (TPP). In certain embodiments, the stabilizer for use in the present disclosure is a non-crosslinked polymer, including, but not limited to, polystyrene. Additional stabilizers contemplated herein include soluble polymers, waxes, triglycerides, and paraffin waxes.

[0109] The stabilizer should form a homogeneous mixture with the sulfonyl oxidizing agent upon evaporation of the solvent. Stabilizers that tend to cause crystallization are not considered suitable. Thus, diphenyl sulfone (DPS) is not suitable since it has been found to form crystals.

[0110] In general, the stabilizer described herein reduces the heat generated during the reaction of a sulfonyl oxidizing agent such as methanesulfonyl azide. The amount of stabilizer relative to the amount of sulfonyl oxidizing agent can be determined. In certain embodiments, the amount of stabilizer provides a composition that can be safely handled and utilized in synthesis. The stabilizer can be in an amount that allows the synthesis of oligonucleotides on a process scale, for example, oligonucleotides can be synthesized in sufficient amounts to perform clinical trials. The stabilizer can be easily removable by solvent or aqueous washing. The stabilizer as used in the synthesis of therapeutic oligonucleotides can be compatible with GMP protocols. In certain embodiments, the stabilizer provides a composition that does not have the risk of explosion upon impact, for example, when methanesulfonyl azide is the sulfonyl oxidizing agent.

[0111] Also provided herein is a stabilized composition comprising methanesulfonyl azide and a stabilizer. In certain embodiments, a stabilized composition is provided that comprises, consists essentially of, or consists of sulfolane and methanesulfonyl azide. The stabilized composition may further comprise a solvent. In certain embodiments, a stabilized composition is provided that comprises, consists essentially of, or consists of sulfolane, methanesulfonyl azide, and optionally acetonitrile. The stabilized composition may optionally be placed in contact with a solid support that carries an oligonucleotide intermediate for use in the synthesis of oligomeric compounds containing phosphoramidate internucleoside linkages as described herein.

[0112] In certain embodiments, the processes described herein are useful for synthesizing oligomeric compounds that comprise or consist of oligonucleotides consisting of linked nucleosides. The present disclosure provides reagents for use in the synthesis of oligonucleotides having any number of modifications as described herein.

[0113] Thus, certain embodiments provide stabilized compositions comprising methanesulfonyl azide and sulfolane, which may comprise 0.1-100 equivalents, 0.1-10 equivalents, 1-10 equivalents, 3-6 equivalents, 4-5 equivalents, 1-2 equivalents, 2-3 equivalents, 3-4 equivalents, 5-6 equivalents, 6-7 equivalents, 7-8 equivalents, 8-9 equivalents, or 9-10 equivalents of sulfolane relative to methanesulfonyl azide, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents of sulfolane relative to methanesulfonyl azide. The concentration of methanesulfonyl azide in the stabilized composition may be from 0.1 to 10M, for example, from 0.5 to 10M, from 0.5 to 5M, from 0.1 to 5M, from 0.3 to 1.5M, from 0.4 to 0.6M, from 0.1 to 0.2M, from 0.2 to 0.3M, from 0.3 to 0.4M, from 0.4 to 0.5M, from 0.4 to 0.6M, from 0.5 to 0.6M, from 0.6 to 0.7M, from 0.7 to 0.8M, from 0.8 to 10M, from 0.9 ... It may be ~0.9M, 0.9-1M, 1-1.1M, 1-1.2M, 1.1-1.2M, 1.1-1.3M, 1.2-1.3M, 1-1.5M, 1.3-1.4M, 1.4-1.5M, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2M. The concentration of sulfolane in the stabilized composition may be 0.1-20M, e.g., 1-20M, 1-10M, 3-6M, 4-5M, 1-2M, 2-3M, 3-4M, 5-6M, 6-7M, 7-8M, 8-9M, or 9-10M, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10M.

[0114] I. Qualification A. Modified Nucleosides Modified nucleosides include stereo-nonstandard nucleosides, or modified sugar moieties, or modified nucleobases, or any combination thereof.

[0115] 1. Certain modified sugar moieties In certain embodiments, the modified sugar moiety is a stereononstandard sugar moiety. In certain embodiments, the sugar moiety is a substituted furanosyl stereostandard sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic furanosyl sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions that correspond to the substitutions of other types of modified sugar moieties.

[0116] a.Sterically non-standard sugar moiety In certain embodiments, the modified sugar moiety is a stereo-nonstandard sugar moiety as shown in Formulas V-XI below: [ka] During the ceremony, One of J1 and J2 is H, and the other of J1 and J2 is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; One of J3 and J4 is H, and the other of J3 and J4 is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; One of J5 and J6 is H, and the other of J5 and J6 is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; One of J7 and J8 is H, and the other of J7 and J8 is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; J9 and J 10 One of J9 and J 10 the other is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; J 11 and J 12 One of them is H and J 11and J 12 the other is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; J 13 and J 14 One of them is H and J 13 and J 14 the other is selected from H, OH, F, OCH3, OCH2CH2OCH3, O-C1-C6 alkoxy, and SCH3; Bx is a heterocyclic base moiety.

[0117] Certain stereononstandard sugar moieties have been previously described, for example, in Seth et al., WO2020 / 072991 and Seth et al., WO2019 / 157531, both of which are incorporated by reference in their entireties.

[0118] b. Substituted stereostandard sugar moiety In certain embodiments, the modified sugar moiety is a substituted stereostandard furanosyl sugar moiety that includes one or more acyclic substituents, including but not limited to, substituents at the 2', 3', 4', and / or 5' positions. In certain embodiments, the furanosyl sugar moiety is a ribosyl sugar moiety. In certain embodiments, the one or more acyclic substituents of the substituted stereostandard sugar moiety are branched. Examples of suitable 2' substituents for substituted stereostandard sugar moieties include, but are not limited to, 2'-F, 2'-OCH3 ("2'-OMe" or "2'-O-methyl"), and 2'-O(CH2)2OCH3 ("2'-MOE"). In certain embodiments, the 2' substituent is halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C2, or 2'-O-C1-C2. 10 Alkoxy, O-C1~C 10 Substituted alkoxy, C1-C 10 Alkyl, C1-C 10 Substituted alkyl, S-alkyl, N(R m )-Alkyl, O-Alkenyl, S-Alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), or OCH2C(=O)-N(R m )(R n )(In the formula, each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10and 2'-substituents described in Cook et al., US 6,531,584, Cook et al., US 5,859,221, and Cook et al., US 6,005,087. Certain embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of 3'-substituents include 3'-methyl (see Frier, et al., The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res., 25, 4429-4443, 1997). Examples of suitable 4' substituents for substituted stereostandard sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO2015 / 106128. Examples of suitable 5' substituents for substituted stereostandard sugar moieties include, but are not limited to, 5'-methyl (R or S), 5'-allyl, 5'-ethyl, 5'-vinyl, and 5'-methoxy. In certain embodiments, non-bicyclic modified sugars contain more than one non-bridging sugar substituent, such as 2'-F-5'-methyl sugar moieties, as well as modified sugar moieties and modified nucleosides described in Migawa et al., WO2008 / 101157 and Rajeev et al., US2013 / 0203836.2',4'-difluoro modified sugar moieties are described in Martinez-Montero, et al., Rigid 2',4'-difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase. J. Org. Chem., 2014, 79:5627-5635. Modified sugar moieties, including 2' modifications (OMe or F) and 4' modifications (OMe or F), are also described in Malek-Adamian, et al., J. Org. Chem, 2018, 83:9839-9849.

[0119] In certain embodiments, the 2'-substituted stereostandard nucleoside is selected from the group consisting of F, NH, N, OCF, OCH, SCH, O(CH)NH, CHCH=CH, OCHCH=CH, OCHCHOCH, O(CH)SCH, O(CH)ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (OCH2C(=O)-N(R m )(R n ))(In the formula, each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 The sugar moiety comprises a non-bridging 2' substituent selected from:

[0120] In certain embodiments, 2'-substituted stereostandard nucleosides comprise a sugar moiety that includes a non-bridging 2'-substituent selected from F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 ("NMA").

[0121] In certain embodiments, a 2'-substituted stereostandard nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, OCH3, and OCH2CH2OCH3.

[0122] In certain embodiments, the 4'O of 2'-deoxyribose may be replaced with S to generate 4'-thio DNA (see Takahashi, et al., Nucleic Acids Research 2009, 37:1353-1362). This modification may be combined with other modifications detailed herein. In certain such embodiments, the sugar moiety is further modified at the 2' position. In certain embodiments, the sugar moiety comprises a 2'-fluoro. A thymidine with this sugar moiety is described in Watts, et al., J. Org. Chem. 2006, 71(3):921-925 (4'-S-fluoro 5-methylarauidine or FAMU).

[0123] C. Bicyclic nucleosides Certain nucleosides contain a modified sugar moiety that includes a bridging sugar substituent that forms a second ring to provide a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a 4' to 2' bridge between the 4' and 2' furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of sugar moieties containing such 4' to 2' bridging sugar substituents include 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CHOCH3)-O-2' ("constrained MOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., US 7,399,845; Bhat et al., US 7,569,686; Swayze et al., US 7,569,686; al., US 7,741,457, and Swayze et al., US 8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., US 8,278,283), 4'-CH2-N(OCH3)-2' and analogs 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, e.g., Zhou, et al., US 7,741,457, and Swayze et al., US 8,022,193), al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see, e.g., Seth et al., US8,278,426), 4'-C(R a R b )-N(R)-O-2',4'-C(R a R b )-ON(R)-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2' (wherein each R, Ra , and R b are independently H, a protecting group, or C1-C 12 Bicyclic sugars including, but not limited to, 4'-C(=O)-N(CH)2-2', 4'-C(=O)-N(R)2-2', 4'-C(=S)-N(R)2-2' and analogs thereof (see, for example, Obika et al., WO2011052436A1, Yusuke's WO2017018360A1).

[0124] The molecular weight of the particles was obtained by analyzing the solvent, Freier et al.,Nucleic Acids Research,1997,25(22),4429-4443;Albaek et al.,J.Org.Chem.,2006,71,7731-7740;Singh et al.,Chem.Commun.,1998,4,455-456;Koshkin et al 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 al., J. Am. Chem. Soc., 2017, 129, 8362-8379; Elayadi et al.,; al.,US6,268,490, Imanishi et al.,US6,770,748,Imanishi et al.,USRE44,779,Wengel et al.,US6,794,499; al.,US8,080,644, Wengel et al.,US8,034,909, Wengel et al.,US8,153,365, Wengel et al.,US7,572,582, and Ramasamy et al.,US6,525,191 al., WO2004 / 106356, Wengel et al., WO1999 / 014226, Seth et al., WO2007 / 134181, Seth et al., US7,547,684, Seth et al., US7,666,854, Seth et al al.,US8,088,746;Seth et al.,US7,750,131;Seth et al.,USSee US Pat. No. 8,030,467, Seth et al., US Pat. No. 8,268,980, Seth et al., US Pat. No. 8,546,556, Seth et al., US Pat. No. 8,530,640, Migawa et al., US Pat. No. 9,012,421, Seth et al., US Pat. No. 8,501,805, as well as Allerson et al., U.S. Patent Publication No. US2008 / 0039618 and Migawa et al., US2015 / 0191727.

[0125] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by their isomeric configuration. For example, LNA nucleosides (described herein) can be in the α-L or β-D configuration. [ka] α-L-methyleneoxy (4'-CH2-O-2') or α-L-LNA bicyclic nucleosides have been incorporated into antisense oligonucleotides and 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. When the position of a particular bicyclic nucleoside (e.g., LNA) is specified in the embodiments illustrated herein, they are in the β-D configuration unless otherwise specified.

[0126] In certain embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (eg, 5'-substituted and 4'-2'-bridging sugars).

[0127] The term "substituted" following a furanosyl ring position, such as "2' substituted" or "2'-4' substituted," indicates that it is the only position or positions in the oligonucleotide that has a substituent other than those found on the unmodified sugar moiety.

[0128] d.Sugar substitutes In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described herein. For example, certain sugar surrogates include a 4' sulfur atom as well as substitutions at the 2' position (see, e.g., Bhat et al., US 7,875,733 and Bhat et al., US 7,939,677) and / or 5' position.

[0129] In certain embodiments, the sugar surrogate comprises a ring having more than five atoms. For example, in certain embodiments, the sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid ("HNA"), altritol nucleic acid ("ANA"), mannitol nucleic acid ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro-HNA ("F-HNA" (see, e.g., Swayze et al., US8,088,904; Swayze et al., US8,440,803; Swayze et al., US8,796,437; and Swayze et al., US9,005,906), which may also be referred to as F-THP or 3'-fluorotetrahydropyran).

[0130] In certain embodiments, the sugar surrogate comprises a ring that does not have any heteroatoms. For example, nucleosides containing bicyclo[3.1.0]-hexane have been described (see, e.g., Marquez, et al., J. Med. Chem. 1996, 39:3739-3749).

[0131] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510, and Summerton et al., US5,698,685, Summerton et al., US5,166,315, Summerton et al., US5,185,444, and Summerton et al., US5,034,506). As used herein, the term "morpholino" refers to a sugar surrogate comprising the following structure: [ka]

[0132] In certain embodiments, morpholinos may be modified, for example, by adding or changing various substituents from the morpholino structure above. Such sugar surrogates are referred to herein as "modified morpholinos." In certain embodiments, the morpholino residue replaces the entire nucleotide, including the internucleoside linkage, and has the structure shown below, where Bx is a heterocyclic base moiety. [ka]

[0133] In certain embodiments, the sugar surrogate comprises an acyclic moiety. Examples of nucleosides and oligonucleotides that comprise such acyclic sugar surrogates include, but are not limited to, peptide nucleic acids ("PNAs"), acyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), glycol nucleic acids ("GNAs", see, Schlegel, et al., J. Am. Chem. Soc. 2017, 139:8537-8546), and the nucleosides and oligonucleotides described in Manoharan et al., WO2011 / 133876. In certain embodiments, the acyclic sugar surrogate comprises: [ka] is selected from.

[0134] Many other bicyclic and tricyclic sugars and sugar surrogate ring systems that can be used in modified nucleosides are known in the art. Certain such ring systems are described in Hanessian, et al., J.Org.Chem., 2013, 78:9051-9063, including bcDNA and tcDNA. Modifications to bcDNA and tcDNA, such as 6'-fluoro, have also been described (Dogovic and Leumann, J.Org.Chem., 2014, 79:1271-1279).

[0135] e. Conjugated nucleosides and terminal groups In certain embodiments, the modified sugar moiety comprises a conjugate group and / or a terminal group. The modified sugar moiety is linked to the conjugate group via a conjugate linker. In certain embodiments, the modified furanosyl sugar moiety comprises a conjugate group attached at the 2', 3', or 5' position. In certain embodiments, the 3'-most sugar moiety of the nucleoside is modified with a conjugate group or terminal group. In certain embodiments, the 5'-most sugar moiety of the nucleoside is modified with a conjugate group or terminal group. In certain embodiments, the sugar moiety near the 3' end of the nucleoside is modified with a conjugate group. In certain embodiments, the sugar moiety near the 5' end of the nucleoside is modified with a conjugate group.

[0136] Examples of terminal groups include, but are not limited to, a conjugate group, a capping group, a phosphate group, a protecting group, a modified or unmodified nucleoside, and two or more nucleosides that are independently modified or unmodified.

[0137] In certain embodiments, the 5'-terminal end group comprises a stabilizing phosphate group. In certain such embodiments, the phosphorus atom of the stabilizing phosphate group is attached to the 5'-terminal nucleoside via a phosphorus-carbon bond. In certain embodiments, the carbon of the phosphorus-carbon bond is in turn attached to the 5'-position of the nucleoside.

[0138] In certain embodiments, the oligonucleotide comprises a 5' stabilized phosphate group having the formula: [ka] During the ceremony, R a and R c are each independently OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino, or substituted amino; R b is O or S, X is substituted or unsubstituted C, where X is attached to the 5'-terminal nucleoside. In certain such embodiments, X is attached to the atom at the 5' position of the 5'-terminal nucleoside. In certain such embodiments, the 5' atom is carbon, and the bond between X and the 5' carbon of the 5'-terminal nucleoside is a carbon-carbon single bond. In certain embodiments, it is a carbon-carbon double bond. In certain embodiments, it is a carbon-carbon triple bond. In certain embodiments, the 5' carbon is substituted. In certain embodiments, X is substituted. In certain embodiments, X is unsubstituted.

[0139] In certain embodiments, the oligonucleotide comprises a 5' stabilized phosphate group having the formula: [ka] During the ceremony, R a and R c are each independently OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino, or substituted amino; R b is O or S, X is substituted or unsubstituted C; Y is selected from C, S, and N. In certain embodiments, Y is substituted or unsubstituted C. The bond between X and Y can be a single bond, a double bond, or a triple bond.

[0140] Certain 5' stabilizing phosphate groups have been previously described, for example, in Prakash et al., WO2011 / 139699 and Prakash et al., WO2011 / 139702, which are hereby incorporated by reference in their entireties.

[0141] In certain embodiments, the stabilizing phosphate group is a 5'-vinylphosphonate or a 5'-cyclopropylphosphonate.

[0142] In certain embodiments, the terminal group at the 5' end is a 5'-mesyl phosphoramidate having formula XII: [ka] In the formula, Z is O or S.

[0143] In certain embodiments, the terminal group at the 5' end is a 5'-mesyl phosphoramidate having formula XIII. [ka]

[0144] 2. Modified Nucleobases In certain embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines, alkyl- or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6 substituted purines. In certain embodiments, modified nucleobases include 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5 -bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp).Modified nucleobases can also include bases in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further nucleobases include those disclosed in Merigan et al., US 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288, and Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443. In certain embodiments, the modified nucleoside comprises a double-headed nucleoside having two nucleobases. Such compounds are described in detail in Sorinas et al., J. Org. Chem, 2014 79:8020-8030.

[0145] Publications that teach the preparation of certain of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, Manoharan et al., US2003 / 0158403, Manoharan et al., US2003 / 0175906, Dinh et al., US4,845,205, Spielvogel et al., US5,130,302, Rogers et al., US5,134,066, Bischofberger et al., US5,175,273, Urdea et al., US5,367,066, Benner et al., US5,432,272, Matteucci et al., US5,434,257, Gmeiner et al., US5,457,187, Cook et al., US5,459,255, Froehler et al., US5,459,255, al.,US5,484,908, Matteucci et al.,US5,502,177, Hawkins et al.,US5,525,711, Haralambidis et al.,US5,552,540, Cook et al.,US5,587,469, Froehler et al.,US5,594,121, Switzer et al. al.,US5,596,091, Cook et al.,US5,614,617, Froehler et al.,US5,645,985, Cook et al.,US5,681,941, Cook et al.,US5,811,534, Cook et al.,US5,750,692, Cook et al. al., US5,948,903, Cook et al. al., US5,587,470, Cook et al., US5,457,191, Matteucci et al., US5,763,588, Froehler et al., US5,830,653, Cook et al., US5,808,027, Cook et al.,6,166,199, and Matteucci et al. al., US6,005,096 included.

[0146] In certain embodiments, the compound comprises or consists of the modified oligonucleotide complementary to the target nucleic acid, and comprises one or more modified nucleobases.In certain embodiments, the modified nucleobase is 5-methylcytosine.In certain embodiments, each cytosine is 5-methylcytosine.

[0147] B. Modified Internucleoside Linkages a. Internucleoside linkage of formula I In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I are selected over compounds lacking such internucleoside linkages having formula I due to one or more desirable properties. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced cellular uptake. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced affinity for target nucleic acids. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have increased stability in the presence of nucleases. In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases. In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced bioavailability. In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced RNase H activity. In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced RNAi activity. In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have enhanced CRISPR activity.In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have reduced interaction with certain proteins. In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified internucleoside linkages having formula I have increased interaction with certain proteins. The methods of making oligonucleotides having at least one internucleoside linkage of formula I (including but not limited to formulas II-IV) may be used to make oligomeric compounds having any of the above properties.

[0148] In certain embodiments, oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprise or consist of modified oligonucleotides complementary to a target nucleic acid, comprising one or more modified internucleoside linkages having formula I: [ka] wherein independently for each internucleoside linkage of Formula I: X is selected from O or S; R is an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C1-C6 alkoxy, a C1-C 20 Alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C 20 It is selected from alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkynyl, and a conjugate group.

[0149] Other internucleoside linkages In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides contain one or more internucleoside linkages of formula I and one or more internucleoside linkages that are not of formula I. In certain embodiments, such internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an oligomeric compound other than at least one internucleoside linkage of formula I is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of an oligomeric compound other than at least one internucleoside linkage of formula I is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. [ka]

[0150] In certain embodiments, the nucleosides of modified oligonucleotides can be linked together using any internucleoside bond.Two major classes of internucleoside bond are defined by the presence or absence of phosphorus atom.Representative phosphorus-containing internucleoside bond includes unmodified phosphodiester internucleoside bond, modified phosphotriester, such as THP phosphotriester and isopropyl phosphotriester, phosphonate, such as methyl phosphonate, isopropyl phosphonate, isobutyl phosphonate, and phosphonoacetate, phosphoramidate, phosphorothioate, and phosphorodithioate ("HS-P=S"). Representative non-phosphorus-containing internucleoside linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (-OC(=O)(NH)-S-); siloxane (-O-SiH2-O-); formacetal, thioacetamide (TANA), alto-thioformacetal, glycinamide, and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to naturally occurring phosphate linkages, modified internucleoside linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. Methods for the preparation of phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those of skill in the art.

[0151] Neutral internucleoside linkages include, but are not limited to, phosphotriester, phosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Further neutral internucleoside linkages include nonionic linkages, including siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH2 component moieties.

[0152] B chiral internucleoside bond 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 prepared as a population of modified oligonucleotides containing sterically random internucleoside linkages or as a population of modified oligonucleotides containing phosphorothioate linkages in a specific stereochemical configuration. In certain embodiments, the population of modified oligonucleotides contains phosphorothioate internucleoside linkages, where all of the phosphorothioate internucleoside linkages are sterically random. Such modified oligonucleotides can be produced using a synthesis method that results in random selection of the stereochemical configuration of each phosphorothioate linkage. All phosphorothioate linkages described herein are sterically random unless otherwise specified. Nevertheless, as will be well understood by those skilled in the art, each individual oligonucleotide molecule of each individual phosphorothioate has a defined stereochemical configuration. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides that contain one or more specific phosphorothioate internucleoside linkages in a specific stereochemical configuration that is independently selected.In certain embodiments, the specific configuration of the specific phosphorothioate linkage is present in at least 65% of the molecules in the population.In certain embodiments, the specific configuration of the specific phosphorothioate linkage is present in at least 70% of the molecules in the population.In certain embodiments, the specific configuration of the specific phosphorothioate linkage is present in at least 80% of the molecules in the population.In certain embodiments, the specific configuration of the specific phosphorothioate linkage is present in at least 90% of the molecules in the population.In certain embodiments, the specific configuration of the specific phosphorothioate linkage is present in at least 99% of the molecules in the population. Such populations of chirally enriched modified oligonucleotides can be generated using synthetic methods known in the art, such as those described in 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 of the indicated phosphorothioates 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, the modified oligonucleotides comprising the (Rp) and / or (Sp) phosphorothioates each comprise one or more of the following formulas, where "B" represents a nucleobase: [ka]

[0153] Unless otherwise specified, the chiral internucleoside linkages of the modified oligonucleotides described herein can be stereo-random or can be in a specific stereochemical configuration.

[0154] In certain embodiments, the internucleoside linkage of formula I may contain a chiral center. In certain embodiments, the modified oligonucleotide contains a chiral linkage of formula II, as shown below. [ka]

[0155] c. Alternative examples of 5' to 3' internucleoside linkages In certain embodiments, the nucleic acids may be linked 2' to 5' rather than the standard 3' to 5' linkage. Such linkages are exemplified below. [ka]

[0156] In certain embodiments, the nucleosides can be linked by 2',3'-phosphodiester bonds. In certain such embodiments, the nucleosides are threofuranosyl nucleosides (TNAs) (see Bala, et al., J Org. Chem. 2017, 82:5910-5916). The TNA bond is shown below: [ka]

[0157] Additional modified linkages include α,β-D-CNA type linkages and related conformationally constrained linkages, as shown below. The synthesis of such molecules has been described previously (Dupouy, et al., Angew. Chem. Int. Ed. Engl., 2014, 45:3623-3627; Borsting, et al. Tetrahedron, 2004, 60:10955-10966; Ostergaard, et al., ACS Chem. Biol. 2014, 9:1975-1979; Dupouy, et al., Eur. J. Org. Chem., 2008, 1285-1294; Martinez, et al., PLoS One, 2011, 6:e25510; Dupouy, et al., Eur. J. Org. Chem., 2007, 5256-5264; Boissonnet, et al., New See J. Chem., 2011, 35:1528-1533). [ka]

[0158] d. Bonds with conjugate groups In certain embodiments, the internucleoside linking group may comprise a conjugate group. In certain embodiments, the internucleoside linking group of formula I comprises a conjugate group. In certain embodiments, the conjugate group of the modified oligonucleotide may be attached to the remainder of the modified oligonucleotide via a modified internucleoside having formula I: [ka] wherein R comprises a conjugate group. In certain embodiments, the conjugate group comprises a cell targeting moiety. In certain embodiments, the conjugate group comprises a carbohydrate or carbohydrate cluster. In certain embodiments, the conjugate group comprises N-acetylgalactosamine (GalNAc). In certain embodiments, the conjugate group comprises a lipid. In certain embodiments, the conjugate group comprises C 10 ~C 20 In certain embodiments, the conjugate group is C 16 Contains alkyl.

[0159] In certain embodiments, the internucleoside linking group that comprises the conjugate group has formula IV. [ka]

[0160] II. A specific motif In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of oligonucleotides. Modified oligonucleotides can be described by their motifs, for example, the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages. In certain embodiments, modified oligonucleotides comprise one or more stereononstandard nucleosides. In certain embodiments, modified oligonucleotides comprise one or more stereostandard nucleosides. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising modified nucleobases. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkages. In such embodiments, the modified, unmodified, and otherwise modified sugar moieties, nucleobases, and / or internucleoside linkages of modified oligonucleotides define a pattern or motif. In certain embodiments, the patterns or motifs of sugar moieties, nucleobases, and internucleoside linkages are each independent of each other. Thus, a modified oligonucleotide can be described by its sugar motif, nucleobase motif, and / or internucleoside linkage motif (as used herein, nucleobase motif describes a modification to a nucleobase independent of the sequence of the nucleobases).

[0161] A. Certain glycomotifs In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of an oligonucleotide. In certain embodiments, the oligonucleotide comprises one or more types of modified sugar and / or unmodified sugar moieties arranged in defined patterns or sugar motifs along the oligonucleotide or a region thereof. In certain instances, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0162] In certain embodiments, the modified oligonucleotide comprises or consists of a gapmer. The sugar motif of the gapmer defines the regions of the gapmer, namely, a 5' region, a central region (gap), and a 3' region. The central region is directly linked to the 5' region and the 3' region without any intervening nucleosides. The central region is a deoxy region. The nucleoside at the first position (position 1) from the 5' end of the central region and the nucleoside at the last position of the central region are adjacent to the 5' region and the 3' region, respectively, and each comprise a sugar moiety independently selected from a 2'-deoxyfuranosyl sugar moiety or a sugar surrogate. In certain embodiments, the nucleoside at position 1 of the central region and the nucleoside at the last position of the central region are DNA nucleosides selected from stereostandard or stereononstandard DNA nucleosides having any of formulas I-VII, where each J is H. In certain embodiments, the nucleosides at the first and last positions of the central region adjacent to the 5' and 3' regions are stereostandard DNA nucleosides. Unlike the nucleosides at the first and last positions of the central region, the nucleosides at other positions within the central region may contain a 2'-substituted furanosyl sugar moiety or a substituted stereononstandard sugar moiety or a bicyclic sugar moiety. In certain embodiments, each nucleoside within the central region supports RNase H cleavage. In certain embodiments, multiple nucleosides within the central region support RNase H cleavage.

[0163] As used herein, the lengths (number of nucleosides) of these three regions of a gapmer may be provided using the notation [number of nucleosides in the 5' region]-[number of nucleosides in the central region]-[number of nucleosides in the 3' region]. Thus, a 3-10-3 gapmer consists of 3 linked nucleosides in each of the 3' and 5' regions, and 10 linked nucleosides in the central region. When such a designation is followed by a specific modification, the modification is of each sugar moiety of each of the 5' and 3' regions, with the central region nucleosides containing a stereostandard DNA sugar moiety. Thus, a 5-10-5 MOE gapmer consists of 5 linked nucleosides each containing a 2'-MOE-stereostandard sugar moiety in the 5' region, 10 linked nucleosides each containing a stereostandard DNA sugar moiety in the central region, and 5 linked nucleosides each containing a 2'-MOE-stereostandard sugar moiety in the 3' region. A 5-10-5 MOE gapmer with a substituted stereononstandard nucleoside at position 2 of the gap has a 10 nucleoside gap in which the second nucleoside of the gap is a substituted stereononstandard nucleoside rather than a stereostandard DNA nucleoside. Such oligonucleotides may also be described as 5-1-1-8-5 MOE / substituted stereononstandard / MOE gapmers. A 3-10-3 cEt gapmer consists of three linked nucleosides each containing a cEt in the 5' region, ten linked nucleosides each containing a stereostandard DNA sugar moiety in the central region, and three linked nucleosides each containing a cEt in the 3' region. A 3-10-3 cEt gapmer with a substituted stereononstandard nucleoside at position 2 of the gap has a 10 nucleoside gap in which the second nucleoside of the gap is a substituted stereononstandard nucleoside rather than a stereostandard DNA nucleoside. Such oligonucleotides may also be described as 3-1-1-8-3 cEt / substituted stereononstandard / cEt gapmers.

[0164] The sugar motif of a 3-10-3 cEt gapmer may also be represented by the notation kkk-d(10)-kkk, where each "k" represents a cEt and each "d" represents a 2'-β-D-deoxyribosyl sugar moiety. This sugar motif is independent of the nucleobase sequence, the internucleoside linkage motif, and any nucleobase modifications. The 5-10-5 MOE gapmer may also be represented by the notation eeeee-d(10)-eeeee or e(5)-d(10)-e(5), where each "e" represents a 2'-MOE-β-D-ribofuranosyl sugar moiety and each "d" represents a 2'-β-D-deoxyribosyl sugar moiety.

[0165] In certain embodiments, each nucleoside of a modified oligonucleotide, or a portion thereof, comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, a sugar surrogate, or a 2'-deoxyribosyl sugar moiety. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from a morpholino, a modified morpholino, a PNA, a THP, and an F-HNA.

[0166] In certain embodiments, the modified oligonucleotide comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleosides that comprise a modified sugar moiety. In certain embodiments, the modified sugar moiety is independently selected from a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from a morpholino, a modified morpholino, THP, and F-HNA.

[0167] In certain embodiments, each nucleoside of a modified oligonucleotide comprises a modified sugar moiety (a "fully modified oligonucleotide"). In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from a morpholino, a modified morpholino, THP, and F-HNA. In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises the same modified sugar moiety (a "uniformly modified sugar motif"). In certain embodiments, the uniformly modified sugar motif is between 7 and 20 nucleosides in length. In certain embodiments, each nucleoside of a uniformly modified sugar motif comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from a morpholino, a modified morpholino, THP, and F-HNA. In certain embodiments, a modified oligonucleotide having at least one fully modified sugar motif may also contain at least one, at least two, at least three, or at least four 2'-deoxyribonucleosides.

[0168] B. Certain nucleobase motifs In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of oligonucleotides. In certain embodiments, the oligonucleotides comprise modified and / or unmodified nucleobases arranged in a defined pattern or motif along the oligonucleotide or a region thereof. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in the modified oligonucleotide are 5-methylcytosine.

[0169] In certain embodiments, the modified oligonucleotide comprises a block of modified nucleobases. In certain such embodiments, the block is at the 3' end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides from 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 from the 5' end of the oligonucleotide.

[0170] In certain embodiments, one nucleoside that comprises modified nucleobase is in the central region of modified oligonucleotide.In certain such embodiments, the sugar moiety of said nucleoside is 2'-β-D-deoxyribosyl moiety.In certain such embodiments, modified nucleobase is selected from 5-methylcytosine, 2-thiopyrimidine, 2-thiothymine, 6-methyladenine, inosine, pseudouracil, or 5-propyne pyrimidine.

[0171] C. Certain internucleoside linkage motifs In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of an oligonucleotide. In certain embodiments, the oligonucleotide comprises modified and / or unmodified internucleoside linkages arranged in a defined pattern or motif along the oligonucleotide or a region thereof. In certain embodiments, one or two of the 5'-most internucleoside linkages are internucleoside linkages of formula I. In certain embodiments, one or two of the 3'-most internucleoside linkages are internucleoside linkages of formula I. In certain embodiments, each internucleoside linkage is selected from an internucleoside linkage of formula I, a phosphorothioate internucleoside linkage, and a phosphodiester internucleoside linkage. In certain embodiments, each internucleoside linkage is selected from an internucleoside linkage of formula I and a phosphodiester internucleoside linkage.

[0172] In certain embodiments, each phosphorothioate internucleoside linkage is independently selected from sterically random phosphorothioates, (Sp) phosphorothioates, and (Rp) phosphorothioates. In certain embodiments, all of the internucleoside linkages in the central region of the modified oligonucleotide are modified. In certain such embodiments, all of the phosphorothioate linkages are sterically random. In certain embodiments, all of the phosphorothioate linkages in the 5' and 3' regions are (Sp) phosphorothioates, and the central region comprises at least one Sp, Sp, Rp motif. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides that comprise such internucleoside linkage motifs.

[0173] In certain embodiments, the double-stranded antisense compound is a double-stranded RNAi compound comprising an RNAi antisense modified oligonucleotide and an RNAi sense modified oligonucleotide, wherein one or both of the RNAi antisense modified oligonucleotide and / or the RNAi sense oligomeric compound have one or more modified internucleoside linkage groups having formula I. In certain embodiments, the RNAi antisense modified oligonucleotide comprises at least two, at least three, at least four, at least five, or at least six modified internucleoside linkage groups having formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises at least two, at least three, at least four, at least five, or at least six modified internucleoside linkage groups having formula I.

[0174] In certain embodiments, the RNAi antisense modified oligonucleotide comprises exactly one modified internucleoside linkage group having formula I. In certain embodiments, the RNAi antisense modified oligonucleotide comprises exactly two modified internucleoside linkage groups having formula I. In certain embodiments, the RNAi antisense modified oligonucleotide comprises exactly three modified internucleoside linkage groups having formula I. In certain embodiments, the RNAi antisense modified oligonucleotide comprises exactly four modified internucleoside linkage groups having formula I.

[0175] In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly one modified internucleoside linking group having formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly two modified internucleoside linking groups having formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly three modified internucleoside linking groups having formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly four modified internucleoside linking groups having formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly five modified internucleoside linking groups having formula I.

[0176] In certain embodiments, at least one of the five 3'-most internucleoside linkage groups of an RNAi antisense modified oligonucleotide is a modified internucleoside linkage group having Formula I. In certain embodiments, at least two of the five 3'-most internucleoside linkage groups of an RNAi antisense modified oligonucleotide are modified internucleoside linkage groups having Formula I.

[0177] D. Certain Modified Oligonucleotides In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of modified oligonucleotides. In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into the modified oligonucleotide. In certain embodiments, the modified oligonucleotides are characterized by their modifications, motifs, and total length. In certain embodiments, each such parameter is independent of the other. Thus, unless otherwise indicated, each internucleoside linkage of a modified oligonucleotide may be modified or unmodified, and may or may not follow the modification pattern of the sugar moiety. Similarly, such modified oligonucleotides may contain one or more modified nucleobases independent of the sugar modification pattern. Furthermore, in certain cases, modified oligonucleotides are described by total length or range and by the length or range of length of two or more regions (e.g., regions of nucleosides with specified sugar modifications), and in such situations, the selection of numbers for each range may result in oligonucleotides whose total length falls outside the specified range. In such situations, both elements must be met. For example, in certain embodiments, a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions or segments A, B, and C, where region or segment A consists of 2-6 linked nucleosides having a specified sugar moiety, region or segment B consists of 6-10 linked nucleosides having a specified sugar moiety, and region or segment C consists of 2-6 linked nucleosides having a specified sugar moiety. In such embodiments, modified oligonucleotides in which A and C are each composed of 6 linked nucleosides and B consists of 10 linked nucleosides are not included (even though those numbers of nucleosides are allowed within the requirements for A, B, and C) because the total length of such oligonucleotide is 22, which exceeds the upper limit of 20 for the total length of the modified oligonucleotide.Unless otherwise indicated, all modifications are independent of the nucleobase sequence, except that in oligonucleotide sequences, modified nucleobase 5-methylcytosine is necessarily "C". In certain embodiments, when DNA nucleosides or DNA-like nucleosides containing T in DNA sequences are replaced with RNA-like nucleosides, nucleobase T is replaced with nucleobase U. Each of these compounds has the same target RNA.

[0178] In certain embodiments, an oligonucleotide consists of X to Y linked nucleosides, where X represents the minimum number of nucleosides within that range and Y represents the maximum number of nucleosides within that range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, provided that X≦Y. For example, in certain embodiments, the oligonucleotides are 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13~18 pieces, 13~19 pieces, 13~20 pieces, 13~21 pieces, 13~22 pieces, 13~23 pieces, 13~24 pieces, 13~25 pieces, 13~26 pieces, 13~27 pieces, 13~28 pieces, 13~29 pieces, 13~30 pieces, 14~15 pieces, 14~16 pieces, 14~17 pieces, 14~18 pieces, 14~19 pieces, 14~20 pieces, 14~21 pieces, 14~22 pieces, 14~23 pieces, 14~24 pieces, 14~25 pieces, 14~26 pieces, 14~27 pieces, 14~28 pieces, 14~29 pieces, 14~30 pieces, 15~16 pieces, 15~17 pieces, 15~18 pieces, 15~19 pieces, 15~20 pieces, 15~21 pieces, 15~22 pieces, 15~23 pieces, 15~24 pieces, 15~25 pieces, 15~26 pieces, 15~27 pieces, 15~28 pieces, 15~29 pieces, 15~30 pieces, 16~17 pieces, 16~18 pieces, 16~19 pieces, 16~20 pieces, 16~21 pieces, 16~22 pieces, 16~23 pieces, 16~24 pieces, 16~25 pieces, 16~26 pieces, 16~27 pieces, 16~28 pieces, 16~29 pieces, 16~30 pieces, 17~18 pieces, 17~19 pieces, 17~20 pieces, 17~21 pieces, 17~22 pieces, 17~23 pieces, 17~24 pieces, 17~25 pieces, 17~26 pieces, 17~27 pieces, 17~28 pieces, 17~29 pieces, 17~30 pieces, 18~19 pieces, 18~20 pieces, 18~21 pieces, 18~22 pieces, 18~23 pieces, 18~24 pieces, 18~25 pieces,18~26 pieces, 18~27 pieces, 18~28 pieces, 18~29 pieces, 18~30 pieces, 19~20 pieces, 19~21 pieces, 19~22 pieces, 19~23 pieces, 1 9~24 pieces, 19~25 pieces, 19~26 pieces, 19~29 pieces, 19~28 pieces, 19~29 pieces, 19~30 pieces, 20~21 pieces, 20~22 pieces, 20 ~23 pieces, 20~24 pieces, 20~25 pieces, 20~26 pieces, 20~27 pieces, 20~28 pieces, 20~29 pieces, 20~30 pieces, 21~22 pieces, 21~ 23 pieces, 21~24 pieces, 21~25 pieces, 21~26 pieces, 21~27 pieces, 21~28 pieces, 21~29 pieces, 21~30 pieces, 22~23 pieces, 22~2 4 pieces, 22~25 pieces, 22~26 pieces, 22~27 pieces, 22~28 pieces, 22~29 pieces, 22~30 pieces, 23~24 pieces, 23~25 pieces, 23~26 pieces, 23~27 pieces, 23~28 pieces, 23~29 pieces, 23~30 pieces, 24~25 pieces, 24~26 pieces, 24~27 pieces, 24~28 pieces, 24~29 pieces , 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 linked nucleosides.

[0179] In certain embodiments, the oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or a specified reference nucleic acid, such as a target nucleic acid. In certain embodiments, a region of the oligonucleotide has a nucleobase sequence that is complementary to a specified reference nucleic acid, such as a second oligonucleotide or a target nucleic acid. In certain embodiments, a region or the entire length of the oligonucleotide has a nucleobase sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to a nucleic acid, such as a second oligonucleotide or a target nucleic acid.

[0180] III. Certain Conjugated Compounds In certain embodiments, the antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of modified oligonucleotides, optionally including a conjugate group. The conjugate group may be attached to either or both ends of the oligonucleotide and / or at any internal position. In certain embodiments, the conjugate group is attached to the 2' position of the nucleoside of the modified oligonucleotide. In certain embodiments, the conjugate group attached to either or both ends of the oligonucleotide is a terminal group. In certain such embodiments, the conjugate moiety or terminal group is attached at the 3' and / or 5' end of the oligonucleotide. In certain such embodiments, the conjugate moiety (or terminal group) is attached at the 3' end of the oligonucleotide. In certain embodiments, the conjugate moiety is attached near the 3' end of the oligonucleotide. In certain embodiments, the conjugate moiety (or terminal group) is attached at the 5' end of the oligonucleotide. In certain embodiments, the conjugate moiety is attached near the 5' end of the oligonucleotide.

[0181] In certain embodiments, at least one internucleoside linkage has formula I: [ka] wherein R comprises a conjugate group. In certain embodiments, R is 16 It is.

[0182] A. Certain Conjugating Groups and Moieties In certain embodiments, modified oligonucleotide comprises one or more conjugate moieties or conjugate groups.In certain embodiments, conjugate groups modify one or more properties of molecule, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance.In certain embodiments, conjugate moieties give new properties to molecule, for example, fluorophores or reporter groups that allow detection of molecule.

[0183] Certain conjugate groups, such as 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. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecane-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or 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, 1999, 20, 533-538). Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid, palmityl 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, i, 923-937), tocopherol groups (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; doi:10.1038 / mtna.2014.72 and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc clusters (e.g., WO2014 / 179620) have been previously described.

[0184] a. Conjugate moiety Conjugate moieties include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0185] In certain embodiments, the conjugate moiety comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazine, chlorothiazide, diazepine, indomethacin, barbiturate, cephalosporin, sulfa drug, antidiabetic drug, antibacterial drug or antibiotic.

[0186] b. Conjugate Linker In certain embodiments, the conjugate group comprises a conjugate linker that connects the conjugate moiety to the remainder of the modified oligonucleotide.In certain embodiments, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is connected to the remainder of the modified oligonucleotide via the conjugate linker by a single bond).In certain embodiments, the conjugate linker comprises a chain structure such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleoside, or amino acid units.

[0187] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0188] In certain embodiments, the conjugate linker, including the conjugate linkers described above, is a bifunctional linking moiety, for example, one known in the art to be useful for attaching a conjugate group to an oligomeric compound, such as an oligonucleotide, provided herein. In general, the bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a specific site on the oligomeric compound, and the other is selected to bind to a conjugate group. Examples of functional groups used in the bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, the bifunctional linking moiety comprises one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0189] 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 C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl, where a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0190] In certain embodiments, the conjugate linker comprises 1-10 linker nucleosides. In certain embodiments, such linker nucleosides are modified nucleosides. In certain embodiments, such linker nucleosides comprise modified sugar moieties. In certain embodiments, the linker nucleosides are unmodified. In certain embodiments, the linker nucleosides comprise an optionally protected heterocyclic base selected from a purine, a substituted purine, a pyrimidine, or a substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. Typically, it is desirable for the linker nucleoside to be 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 via cleavable bonds, hi certain embodiments, such cleavable bonds are phosphodiester bonds.

[0191] Unless otherwise indicated, the conjugate linker comprises 10 or less linker nucleosides. In certain embodiments, the conjugate linker comprises 5 or less linker nucleosides. In certain embodiments, the conjugate linker comprises 3 or less linker nucleosides. In certain embodiments, the conjugate linker comprises 2 or less linker nucleosides. In certain embodiments, the conjugate linker comprises only 1 linker nucleoside.

[0192] In certain embodiments, it is desirable that the conjugate group or conjugate moiety is cleaved from the remainder of the oligonucleotide. For example, in certain situations, oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) or modified oligonucleotides that contain certain conjugate moieties should be taken up into certain cell types, but once the compound is taken up, it is desirable that the conjugate group is cleaved to release the unconjugated oligonucleotide. Thus, certain conjugate moieties may contain one or more cleavable moieties, typically in the conjugate linker. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety is an atomic group that includes at least one cleavable bond. In certain embodiments, the cleavable moiety includes an atomic group that has one, two, three, four, or more than four cleavable bonds. In certain embodiments, the cleavable moiety is selectively cleaved inside the cell or in an intracellular compartment, such as a lysosome. In certain embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme, such as a nuclease.

[0193] In certain embodiments, the cleavable bond is selected from among amide, ester, ether, one or both esters of phosphodiester, phosphate ester, carbamate, or disulfide. In certain embodiments, the cleavable bond is one or both esters of phosphodiester. In certain embodiments, the cleavable moiety comprises a phosphate or a phosphodiester. In certain embodiments, the cleavable moiety is a phosphate or a phosphodiester bond between the oligonucleotide and the conjugate moiety or the conjugate group.

[0194] In certain embodiments, the 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 via a cleavable bond. In certain embodiments, such cleavable bond is an unmodified phosphodiester bond. In certain embodiments, the cleavable moiety is a nucleoside that includes a 2'-deoxyfuranosyl that is linked to either the 3'-terminal nucleoside or the 5'-terminal nucleoside of the oligonucleotide by a phosphodiester internucleoside bond and covalently linked to the remainder of the conjugate linker or conjugate moiety by a phosphodiester or phosphorothioate bond. In certain such embodiments, the cleavable moiety is a nucleoside that includes a 2'-β-D-deoxyribosyl sugar moiety. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.

[0195] c. A particular cell-targeting conjugate moiety In certain embodiments, the conjugate group comprises a cell-targeting conjugate moiety. In certain embodiments, the conjugate group has the general formula: [ka] In the formula, n is 1 to about 3; when n is 1, m is 0; when n is 2 or more, m is 1; j is 1 or 0; and k is 1 or 0.

[0196] In certain embodiments, n is 1, j is 1, and k is 0. In certain embodiments, n is 1, j is 0, and k is 1. In certain embodiments, n is 1, j is 1, and k is 1. In certain embodiments, n is 1, j is 1, and k is 1. In certain embodiments, n is 2, j is 1, and k is 0. In certain embodiments, n is 2, j is 0, and k is 1. In certain embodiments, n is 2, j is 1, and k is 1. In certain embodiments, n is 3, j is 1, and k is 0. In certain embodiments, n is 3, j is 0, and k is 1. In certain embodiments, n is 3, j is 1, and k is 1.

[0197] In certain embodiments, the conjugate group comprises a cell targeting moiety having at least one tethered ligand. In certain embodiments, the cell targeting moiety comprises two tethered ligands covalently attached to the branching group. In certain embodiments, the cell targeting moiety comprises three tethered ligands covalently attached to the branching group.

[0198] In certain embodiments, the cell targeting moiety comprises a branched group comprising one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the branched group comprises a branched aliphatic group comprising a group selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl, amino, oxo, amide, and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl, amino, and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl and ether groups. In certain embodiments, the branched group comprises a monocyclic or polycyclic ring system.

[0199] In certain embodiments, each tether of the cell targeting moiety comprises one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amino, oxo, amide, phosphodiester, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, amino, and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or a neutral linking group. In certain embodiments, each tether comprises a chain of about 6 to about 20 atoms in length. In certain embodiments, each tether comprises a chain of about 10 to about 18 atoms in length. In certain embodiments, each tether comprises a chain length of about 10 atoms.

[0200] In certain embodiments, each ligand of the cell targeting moiety has affinity for at least one receptor type on the target cell. In certain embodiments, each ligand has affinity for at least one receptor type on the surface of a mammalian lung cell.

[0201] In certain embodiments, the cell targeting moiety has affinity for the asialoglycoprotein receptor (ASGPR). In certain embodiments, each ligand of the cell targeting moiety is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a polysaccharide, a modified polysaccharide, or a polysaccharide derivative. In certain such embodiments, the conjugate group comprises a carbohydrate cluster (see, for example, Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting," Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor," J.Med.Chem. 2004, 47, 5798-5808, which are incorporated herein by reference in their entirety). In certain such embodiments, each ligand is an amino sugar or a thio sugar, for example, the amino sugar may be selected from any number of compounds known in the art, such as sialic acid, α-D-galactosamine, β-muramic acid, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-glycolyl-α-neuraminic acid. For example, the thiosugar may be selected from 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

[0202] In certain embodiments, the oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) or modified oligonucleotides described herein comprise a conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem,1995,38,1538-1546, Valentijn et al.,Tetrahedron,1997,53,759-770, Kim et al.,Tetrahedron Lett,1997,38,3487-3490, Lee et al.,Bioconjug Chem,1997,8,762-765, Kato et al. al., Glycobiol,2001,11,821-829, Rensen et al., J Biol Chem,2001,276,37577-37584, Lee et al.,Methods Enzymol,2003,362,38-43, Westerlind et al.,Glycoconj J,2004,21,227-241,Lee et al.,Bioorg Med Chem Lett,2006,16(19),5132-5135, Maierhofer et al.,Bioorg Med Chem,2007,15,7661-7676, Khorev et al.,Bioorg Med Chem,2008,16,5216-5231, Lee et al.,Bioorg Med Chem,2011,19,2494-2500,Kornilova et al.,Analyt Biochem,2012,425,43-46,Pujol et al.,Angew Chemie Int Ed Engl,2012,51,7445-7448、Biessen et al.,J Med Chem,1995,38,1846-1852、Sliedregt et al.,J Med Chem,1999,42,609-618、Rensen et al.,J Med. Chem,2004,47,5798-5808、Rensen et al.,Arterioscler Thromb Vasc Biol,2006,26,169-175、van Rossenberg et al.,Gene Ther,2004,11,457-464、JSato et al.,Am Chem. Soc,2004,126,14013-14022、Lee et al.,J Org Chem,2012,77,7564-7571、Biessen et al.,FASEB J,2000,14,1784-1792、Rajur et al.,Bioconjug Chem,1997,8,935-940、Duff et al.,Methods Enzymol,2000,313,297-321、Maier et al.,Bioconjug Chem,2003,14,18-29、Jayaprakash et al.,Org Lett,2010,12,5410-5413、Manoharan,Antisense Nucleic Acid Drug Dev,2002,12,103-128、Merwin et al.,Bioconjug Chem,1994,5,612-620、Tomiya et al.,Chem Bioorg,20135275-5281, International Application No. WO1998 / 013381, WO2011 / 038356, WO1997 / 046098, WO2008 / 098788, WO2004 / 101619, WO2012 / 037254, WO2011 / 120053, WO2011 / 100131, WO2011 / 163121, WO2012 / 177947, WO2013 / 033230, WO2013 / 075035, WO2012 / 0 83185, WO2012 / 083046, WO2009 / 082607, WO2009 / 134487, WO2010 / 144740, WO2010 / 148013, WO1997 / 020563, WO2010 / 088537, WO2002 / 043771, WO2010 / 129709, WO2012 / 068187, WO2009 / 126933, WO2004 / 024757, WO2010 / 054406, WO201 2 / 089352, WO2012 / 089602, WO2013 / 166121, WO2013 / 165816, U.S. Patent Nos. 4,751,219, 8,552,163, 6,908,903, 7,262,177, 5,994,517, 6,300,319, 8,106,022, 7,491,805, 7,491,805, 7,582,744, 8,137,695, 6,383 ,812, No. 6,525,031, No. 6,660,720, No. 7,723,509, No. 8,541,548, No. 8,344,125, No. 8,313,772, No. 8,349,308, No. 8,450 ,467, No. 8,501,930, No. 8,158,601, No. 7,262,177, No. 6,906,182, No. 6,620,916, No. 8,435,491, No. 8,404,862, No. 7,851,615, Published U.S. Patent Application Publication Nos. US2011 / 0097264, US2011 / 0097265, US2013 / 0004427, US2005 / 0164235, US2006 / 0148740, US2008 / 0281044, US2010 / 0240730, US2003 / 0119724, US2006 / 0183886, US2008 / 0206869, US2011 / 0269814, US2009 / 0286973, US2011 / 0207799, US2012 / 0136042, US2012 / 016539 3, US2008 / 0281041, US2009 / 0203135, US2012 / 0035115, US2012 / 0095075, US2012 / 0101148, US2012 / 0128760, US2012 / 0157509, US2012 / 0230938, US2013 / 0109817, US2013 / 0121954, US2013 / 0178512, US2013 / 0236968, US2011 / 0123520, US2003 / 0077829, US2008 / 0108801, and US2009 / 0203132.

[0203] In certain embodiments, the conjugate group comprises N-acetylgalactosamine (GalNAc).

[0204] In certain embodiments, the conjugate group is attached to the first modified oligonucleotide at the 5' end of the first modified oligonucleotide. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide at the 3' end of the first modified oligonucleotide.

[0205] In certain embodiments, the conjugate group comprises a cell targeting moiety having affinity for the transferrin receptor (TfR), also known as TfR1 and CD71. In certain embodiments, the conjugate group comprises an anti-TfR1 antibody or a fragment thereof. In certain embodiments, the conjugate group comprises a peptide capable of binding to TfR1. In certain embodiments, the conjugate group comprises an aptamer capable of binding to TfR1.

[0206] Compositions and methods for formulating pharmaceutical compositions The antisense agents, oligomeric compounds, and modified oligonucleotides described herein may be mixed with pharma- ceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions. The composition and method of formulating the pharmaceutical composition depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0207] Certain embodiments provide pharmaceutical compositions comprising one or more oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) or salts thereof. In certain such embodiments, the pharmaceutical composition comprises a suitable pharma- ceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises a sterile saline solution and one or more oligomeric compounds. In certain embodiments, such pharmaceutical compositions consist of a sterile saline solution and one or more oligomeric compounds. In certain embodiments, the sterile saline solution is pharmaceutical grade saline. In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and sterile water. In certain embodiments, the pharmaceutical composition consists of an oligomeric compound 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, the pharmaceutical composition consists of one or more oligomeric compounds and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS. The composition and method of formulating a pharmaceutical composition will depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dosage to be administered.

[0208] The oligomeric compounds described herein that are complementary to a target nucleic acid can be utilized in a pharmaceutical composition by combining the oligomeric compound with a suitable pharma- ceutically acceptable diluent or carrier and / or additional components such that the pharmaceutical composition is suitable for injection. In certain embodiments, the pharma- ceutically acceptable diluent is phosphate-buffered saline. Thus, in one embodiment, the method described herein uses a pharmaceutical composition that includes an oligomeric compound complementary to a target nucleic acid and a pharma- ceutically acceptable diluent. In certain embodiments, the pharma- ceutically acceptable diluent is phosphate-buffered saline. In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide provided herein.

[0209] Pharmaceutical compositions comprising the oligomeric compounds provided herein (including oligomeric compounds that are antisense agents or portions thereof) include any pharmaceutically acceptable salts, esters or salts of such esters, or any other oligonucleotides that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans. In certain embodiments, the oligomeric compounds comprise or consist of modified oligonucleotides. Thus, for example, the present disclosure is also directed to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0210] Target Nucleic Acids, Target Regions, and Nucleotide Sequences In certain embodiments, the antisense agent, oligomeric compound, or modified oligonucleotide described herein comprises or consists of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from an mRNA and a pre-mRNA, comprising an intron region, an exon region, and an untranslated region. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the pre-mRNA and the corresponding mRNA are both target nucleic acids of a single compound. In certain such embodiments, the target region is entirely within an intron of the target pre-mRNA. 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 a microRNA. In certain embodiments, the target region is in the 5'UTR of a gene. In certain embodiments, the target region is within a translational repression element region of the target nucleic acid.

[0211] Certain compounds Certain compounds described herein (e.g., antisense agents, oligomeric compounds, and modified oligonucleotides) have one or more asymmetric centers, thus giving rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), α or β, as for sugar anomers, or (D) or (L), as for amino acids. Compounds provided herein that are depicted or described as having a particular stereoisomeric configuration include only the compounds shown. Compounds provided herein that are depicted or described with undefined stereochemical structure include all such possible isomers, including their stereo-random and optically pure forms. All tautomeric forms of the compounds provided herein are included unless otherwise indicated.

[0212] The compounds described herein include variations in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated elements. For example, compounds herein containing hydrogen atoms include 1 All possible deuterium substitutions are included for each H hydrogen atom. Isotopic substitutions encompassed by the compounds herein include: 1 Replaces H. 2 H or 3 H, 12 Replaces C 13 C or 14 C. 14 Replaces N 15 N, 16 Replaces O. 17 O or 18 O, and 32 Replaces S. 33 S, 34 S, 35 S, or 36 These include, but are not limited to, S. In certain embodiments, the substitution of non-radioactive isotopes can provide the oligomeric compounds with new properties that are beneficial for use as therapeutic or research tools. In certain embodiments, the substitution of radioactive isotopes can make the compounds suitable for research or diagnostic purposes, such as imaging. EXAMPLES

[0213] The following examples are intended to illustrate certain aspects of the invention and are not intended to limit the invention in any way.

[0214] Example 1: Preparation of the oxidizing solution Five oxidizing solutions were prepared, each containing the sulfonyl oxidizing agent methanesulfonyl azide (mesyl azide, MsN3). One oxidizing solution contained no stabilizer (control solution) and four oxidizing solutions each contained a stabilizer. The stabilizers tested were triphenyl phosphate (TPP) and diphenyl sulfone (DPS). [ka] The use of diphenyl sulfone is not recommended as crystalline material has been observed to form.

[0215] Solutions were prepared as described below and at the concentrations specified in Table 1. All solutions were stored at 5°C. [Table 1]

[0216] Preparation of 1.0 M MsN3 in acetonitrile [ka] NaN3 (5.4 g, 83 mmol, 1 equiv) was suspended in anhydrous acetonitrile (MeCN, 80 mL) and cooled to 0° C. with stirring under nitrogen. Mesyl chloride (10 g, 6.75 mL, 88.3 mmol, 1.05 equiv) was added dropwise and the reaction was allowed to warm to room temperature over 3 hours. The reaction was filtered to remove insoluble salts to give the desired 1.0 M solution of MsN3 in MeCN.

[0217] Preparation of oxidizing solution 1 20 mL of toluene and 20 mL of 1.0 M MsN3 in MeCN (described above) were added to a 50 mL amber bottle. The bottle was covered with parafilm and capped. The solution was mixed by swirling by hand.

[0218] Preparation of oxidizing solution 2 10 mL of 2.0 M TPP in toluene and 10 mL of 1.0 M MsN3 in MeCN (described above) were added to a 50 mL amber bottle. The bottle was covered with parafilm and capped. The solution was mixed by swirling by hand.

[0219] Example 2: Thermodynamic analysis of the oxidizing solutions The oxidizing solutions 1-3 described above were tested by Dekra (dekra.us / process-safety) using a standard protocol. Briefly, the solvent was removed from each oxidizing solution and the corresponding residues were evaluated by Differential Scanning Calorimetry (DSC) to determine their heat of composition. This test determines the onset temperature of any energy events and the total energy associated with those events. Each event is listed on a separate line in Table 2 below and is labeled as to whether there was a positive (endothermic) or negative (exothermic) change in the enthalpy of the residue as it was heated. 300 J.g -1 The decomposition energy of over 500 J.g indicates a high energy material. -1 The decomposition energy above 100 indicates that the material may have explosive properties.

[0220] A 0.400 mL aliquot of each solution was allowed to evaporate overnight at room temperature to produce a residue that was used in the DSC test. The test samples were loaded into sealed high pressure gold crucibles. An empty crucible of the same type was used as a reference. The sample and reference crucibles were placed in a Mettler Toledo DSC 3+ furnace, which was heated to a starting temperature of 25° C. Once the crucibles were equilibrated with the furnace, they were heated to 400° C. at a constant rate of 5° C. / min. The heat flow from the sample and reference crucibles was recorded throughout the test. Any exothermic activity within the sample will result in a greater heat flow from the sample crucible compared to the reference crucible. The start, end, and peak temperatures were recorded and are provided in Table 2. [Table 2]

[0221] As shown above, the residue containing TPP (Oxidation Solution 2) has a lower combustion energy than the residue from the control solution (Oxidation Solution 1) that does not contain any solid stabilizer.

[0222] Example 3: Synthesis of modified oligonucleotides containing mesyl phosphoramidate internucleoside linkages via oxidative mesylation using mesyl azide solution The diagram below shows a general overview scheme of oligonucleotide synthesis using mesyl azide as an oxidizing agent. An oligonucleotide intermediate, shown as a black and white ribbon, is attached to a solid support (shown as a circle). A phosphoramidite monomer is introduced onto the oligonucleotide using standard techniques. "B(pg)" in the diagram below represents a variable nucleobase with a general protecting group(s). The use of mesyl azide as an oxidizing agent in the second general step results in the oxidation of the phosphotriester bond to give a mesyl phosphoramidate internucleoside bond. [ka] Modified oligonucleotides containing mesyl phosphoramidate internucleoside linkages were prepared using the oxidation solution described in Example 1.

[0223] Oligonucleotide intermediate Compound A was synthesized using standard techniques. Compound A is a 13 nucleoside long modified oligonucleotide intermediate having the nucleobase sequence (5' to 3'): TGGTTATGACTCA (SEQ ID NO: 1). The sugar motif of Compound A is (5' to 3'): ddddddddeeeee, where each "d" represents a 2'-β-D-deoxyribosyl sugar moiety and each "e" represents a 2'-MOE sugar moiety. The internucleoside linkage motif of Compound A is (5' to 3'): sssssssssss, where each "s" represents a phosphorothioate internucleoside linkage. Each cytosine residue is a 5-methylcytosine. The 5'-OH of Compound A is capped with a dimethoxytrityl (DMT) protecting group. The linked nucleosides of Compound A are attached to a solid support.

[0224] Two 2'-β-D-deoxyribosylthymidine nucleosides were linked to compound A via mesyl phosphoramidate internucleoside linkages. Modified oligonucleotides were synthesized on an AKTA Oligopilot 10 (35 μmol scale). Deoxy T phosphoramidites were dissolved in 1:1 MeCN / toluene (v / v) and dried over molecular sieves. The DMT protecting group was removed from the modified oligonucleotide intermediate using 15% DCA in toluene. Deoxy T phosphoramidites were coupled using 3 equivalents of amidite and 10 equivalents of activator (1M 4,5-dicyanoimidazole and 0.1M N-methylimidazole in acetonitrile) per amidite, and the coupling solution was recirculated for 6 min. After flushing with MeCN, oxidation solution containing MsN3 was added (25 equivalents) and recirculated for 25 min, followed by washing with MeCN. After washing, the reaction mixture was treated with 20% acetic anhydride in MeCN (Cap A) and N-methylimidazole in MeCN / pyridine (2:5:3 v / v / v, Cap B) to cap any unsuccessful couplings. The cycle was repeated to incorporate a second mesyl-linked thymidine nucleoside.

[0225] The modified oligonucleotide intermediate, Compound B, was cleaved from the solid support and deprotected using standard techniques to obtain the final modified oligonucleotide. Compound B is a 15 nucleoside long modified oligonucleotide having the sequence (5' to 3'): TTTGGTTATGACTCA (SEQ ID NO: 2). The sugar motif of Compound B is (5' to 3'): ddddddddddeeeee, where each "d" represents a 2'-β-D-deoxyribosyl sugar moiety and each "e" represents a 2'-MOE sugar moiety. The internucleoside linkage motif of Compound B is (5' to 3'): zzsssssssssss, where each "s" represents a phosphorothioate internucleoside linkage and each "z" represents a mesyl phosphoramidate internucleoside linkage. Each cytosine residue is a 5-methylcytosine.

[0226] Compound B was synthesized separately using oxidation solutions 1-5, respectively, resulting in products similarly labeled as compounds B1, B2, B3, B4, and B5. Compounds B1-B5 were analyzed by UV chromatography and liquid chromatography-mass spectrometry. In the oxidative mesylation step, oxidation solution 2 (TPP in 1:1 toluene to MeCN) resulted in completion of the reaction and acceptable purity compared to compound B1 (synthesized using a control solution). In oxidation solutions 2, 3, and 4, the stabilizers did not appear to have any adverse effect on the coupling and may reduce the hazardous risk of handling mesyl azide.

[0227] Example 4: Preparation of a solution of methanesulfonyl azide in acetonitrile with sulfolane as a stabilizer An oxidation solution was prepared containing the sulfonyl oxidizing agent MsN3 in acetonitrile with sulfolane as a stabilizer. The structure of sulfolane is shown below. [ka]

[0228] The solutions were prepared using two methods described below.

[0229] Method 1 A 1000 mL three-neck round bottom flask equipped with an overhead stirrer (Teflon moon impeller, glass shaft) under N2 was charged with NaN3 (70.58 g, 1.09 mol) and dry MeCN (517 mL). Stirring was started at room temperature on an IKA overhead stirrer at a setting of about 2.5 for about 5 minutes before the flask was immersed in an ice bath. Mesyl chloride (80.0 mL, 1.03 mol) was added dropwise via an addition funnel over a period of about 30 minutes. Upon complete addition of the mesyl chloride, the reaction was removed from the ice bath and allowed to stir at room temperature overnight.

[0230] 1The reaction was confirmed to be complete based on the absence of a chemical shift for mesyl chloride (expected value: 3.8 ppm) by H NMR. The concentration of MsN3 was determined to be 2.082 M by quantitative NMR using ethylene carbonate as the analytical standard.

[0231] The reaction was then filtered through a bottle-top filter into a tared polycoated glass bottle equipped with a Teflon-coated magnetic stir bar. The filter cake was rinsed with a small amount of MeCN (approximately 10 mL). Molten sulfolane (621.28 g, 5.17 mol) was then charged to the bottle. The mixture was stirred and the mass and density of the solution were determined and used to dilute the solution with MeCN to a total volume of 1034 mL.

[0232] Method 2 A 1000 mL three-neck round bottom flask equipped with an overhead stirrer (Teflon moon impeller, glass shaft) under N2 was charged with NaN3 (35.29 g, 543 mmol), molten sulfolane (246 g, 2.59 mol), and dry MeCN (270 mL). Stirring was started at room temperature on an IKA overhead stirrer at a setting of about 2.5 for about 5 minutes before the flask was immersed in an ice bath. Mesyl chloride (40.0 mL, 517 mmol) was added dropwise via the addition funnel over a period of about 30 minutes. Upon complete addition of the mesyl chloride, the reaction was removed from the ice bath and allowed to stir at room temperature overnight.

[0233] 1 The reaction was confirmed to be complete based on the absence of a chemical shift for mesyl chloride (expected value: 3.8 ppm) by H NMR. The concentration of MsN3 was determined to be 0.943 M in the reaction mixture by quantitative NMR using ethylene carbonate as the analytical standard.

[0234] The reactions were then filtered through bottle-top filters into tared polycoated glass bottles and stored without further dilution.

[0235] Example 5: Synthesis of modified oligonucleotides containing mesyl phosphoramidate internucleoside linkages via oxidative mesylation using mesyl azide solution Modified oligonucleotides containing mesyl phosphoramidate internucleoside linkages were prepared using a solution of MsN3 in acetonitrile containing sulfolane.

[0236] Modified oligonucleotides were synthesized on an AKTA Oligopilot 10 (40 μmol scale) using a polystyrene-based NittoPhase HL UnyLinker support (405 μmol / g). Fully protected nucleoside phosphoramidites were incorporated using standard solid-phase modified oligonucleotide synthesis conditions described above in Example 3 herein. DNA amidites were dissolved at 0.1 M in 1:1 MeCN / toluene and incorporated using a 6 min recycling time. 1 M 4,5-dicyanoimidazole and 0.1 M N-methylimidazole in MeCN were used as activators. 15% dichloroacetic acid in toluene was used to remove the DMT protecting group. 20% acetic anhydride in MeCN and N-methylimidazole / pyridine / MeCN (20:30:50) were used to cap unsuccessful couplings.

[0237] Oxidation of P(III) species was carried out as follows: 0.05 M iodine in pyridine / HO (9:1) for phosphodiester linkages; or 0.1 M xanthan hydride in 1:1 pyridine:MeCN for phosphorothioate linkages. For incorporation of mesyl phosphoramidate linkages, the modified oligonucleotide intermediate was treated with 0.65 M MsN3 in 1:1 MeCN:sulfolane or 0.65 M MsN3 in MeCN and recirculated for 25 min.

[0238] After termination of the synthesis, the cyanoethyl protecting group was removed using 20% ​​diethylamine in toluene, and the remaining protecting group was cleaved by suspending the solid support in concentrated aqueous ammonia and heating at 55° C. for 14 hours. The support was removed by filtration, and the crude mixture was purified by HPLC using a combined purification, detritylation, and desalting method. During the basic RSR (reversed phase, SAX, reversed phase) method, the sample was loaded onto a RP column (DuPont XT30) in H2O. Elution of the failures was then performed on the RP column with 1:1 (A: 80% MeOH / water, B: 2.5 M NaCl, 50 mM NaOH). DMT cleavage was then performed on the RP column with 6% DCA, followed by washing with water. The detritylated compound was then loaded onto a SAX column with 80% MeOH. The RP column was equilibrated with 50 mM NaOH. A SAX gradient was then run from 0 to 50% with A and B buffers (A: 50 mM NaOH, B: 50 mM NaOH, 2.5 M NaCl). Once the UV absorbance threshold was reached, the compound was then loaded back onto the RP column. NaCl (250 mM) was flushed through the RP column for cation exchange, water was run through the column for desalting, and the final compound was eluted with 1:1 MeCN:water.

[0239] Comparison of product purity after synthesis with and without sulfolane Using standard techniques described herein above, modified oligonucleotide compound 1633475 was synthesized with and without sulfolane and each lot was analyzed for lot purity.

[0240] Compound 1633475 is a 16 nucleoside long modified oligonucleotide intermediate having the nucleobase sequence (5' to 3'): GCATGTTCTCACATTA (SEQ ID NO:3). The sugar motif of compound 1633475 is (5' to 3'): kkkddddddddddkkk, where each "d" represents a 2'-β-D-deoxyribosyl sugar moiety and each "k" represents a cEt sugar moiety. The internucleoside linkage motif of compound 1633475 is (5' to 3'): ssszzzzsssssss, where each "s" represents a phosphorothioate internucleoside linkage and each "z" represents a mesyl phosphoramidate internucleoside linkage. Each cytosine residue is a 5-methylcytosine. Compound 1633475 further contains a 3THAGNhp moiety conjugated to the 3' terminal oxygen of the modified oligonucleotide via a phosphodiester bond, as shown below. [ka]

[0241] Samples of each modified oligonucleotide lot were made up in 0.01% triethylamine in H2O at a concentration of approximately 1 mg / mL. Samples were analyzed by ion-pair HPLC / mass spectrometry (IP-HPLC / MS) on an Agilent 1200 series equipped with a binary pump, online degasser, heated column chamber, autosampler, and multi-wavelength UV detector interfaced to an electrospray mass spectrometer. Analysis was performed using a Waters (Milford, MA, USA) XBridge™ HPLC column (18C, 3.5 μm, 2.1×150 mm, Waters P / N 186003023). A linear gradient of 5 mM tributylammonium acetate and 1 μM EDTA in 10% acetonitrile (mobile phase A) and 5 mM tributylammonium acetate and 1 μM EDTA in 80% acetonitrile (mobile phase B) was used as described in the table below. [Table 3]

[0242] The UV absorbance of the column eluate was measured at 260 nm with a reference wavelength of 400 nm. The column eluate was directly introduced into the ESI-MS. The ESI source was operated in negative mode with a scanning mass signal (m / z) range of (full length product mass) / 4±150.0. Capillary voltage=4000 V, drying gas temperature=260° C., drying gas flow rate=12 L / min, nebulizer pressure=25 psi, fragmentor voltage=100 V.

[0243] To calculate UV purity, the UV peaks of the full-length product (main UV peak), early eluting impurities, and late eluting impurities at 260 nm were identified and integrated in OpenLab ChemStation version C.01.09. The area of ​​the main UV peak was normalized to the sum of the areas of all peaks at 260 nm, which is presented in the table below as UV Purity (%). To calculate MS Purity, the m / z of the full-length product and the m / z of all impurities were identified within the main UV peak. Ion chromatograms for each component mass signal were extracted and integrated. The area of ​​the full-length product signal was normalized to the sum of the component signals, which is presented below as MS Purity (%). Each table represents a different analysis.

[0244] Synthesis of compound 1633475 using 0.65 M MsN3 in MeCN with or without 1.5 M sulfolane produced modified oligonucleotides of similar quality. UV and MS purities are provided in Table 4. The synthesis conditions used were the same except for the solvent in which the MsN3 oxidizing solution was held. [Table 4]

[0245] Example 6: Thermodynamic analysis of mesyl azide in the presence of sulfolane The sulfolane stabilized oxidizing solution MsN3 was tested by Nalas Engineering (nalasengineering.com / process-scale-up) using standard DSC protocols.

[0246] A portion of each solution was allowed to evaporate overnight at room temperature to yield a residue that was used in the DSC test. Analysis was performed using a DSC25 (Waters Instruments). Test samples were loaded into sealed high pressure gold crucibles. An empty crucible of the same type was used as a reference. The sample and reference crucibles were placed in a furnace, which was heated to a starting temperature of 30°C. Once the crucibles were equilibrated with the furnace, they were heated to 500°C at a constant rate of 5°C / min. Heat flow from the sample and reference crucibles was recorded throughout the test and analyzed using TRIOS software. Results are presented in the table below as energy flow, with events presented as exothermic (negative change in enthalpy) or endothermic (positive change in enthalpy). 300 Jg -1 The decomposition energy of over 500 Jg indicates a high-energy material. -1 The decomposition energies above 1000 mV suggest that the material may have explosive properties. As shown in the table below, the residue containing sulfolane has a lower combustion energy than pure MsN3. [Table 5]

[0247] Example 7: Shock Sensitivity Analysis of Mesyl Azide in the Presence of Sulfolane Oxidized solutions of MsN3 stabilized with sulfolane were tested by Nalas Engineering (nalasengineering.com / process-scale-up) using a standard protocol for impact sensitivity. Briefly, samples of material were placed in a BAM Fall Hammer Apparatus and impacted with various amounts of energy. To determine the limiting impact energy, the samples were observed for ignition, flame, or explosion. Observations of MsN3 with sulfolane and pure MsN3 are presented in the table below.

[0248] Impact testing has shown that an approximately 1:1 (w / w) mixture of MsN3:sulfolane appears to mitigate the explosive properties of MsN3.

Table 6

Claims

1. A method for preparing a modified oligonucleotide, comprising contacting a first oligonucleotide intermediate having a phosphite triester nucleoside internucleoside linkage with an oxidation solution containing at least one stabilizer and a sulfonyl oxidizing agent to form a second oligonucleotide intermediate having an internucleoside linking group of formula XIV, 【Chemical 1】 wherein, R is selected from aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, diazol, substituted diazol, C １ ~C ６ alkoxy, C １ ~C ２０ alkyl, C １ ~C ６ alkenyl, C １ ~C ６ alkynyl, substituted C １ ~C ２０ alkyl, substituted C １ ~C ６ alkenyl, substituted C １ ~C ６ alkynyl, and conjugate groups wherein the stabilizer is selected from sulfolane, naphthalene, triphenyl phosphate (TPP), uncrosslinked polymer, polystyrene, soluble polymer, wax, triglyceride, and paraffin wax, said method.

2. R is methyl and the sulfonyl oxidizing agent is methanesulfonyl azide (MsN ３ )), the method according to claim 1.

3. The method according to claim 1, wherein the oxidation solution contains the at least one stabilizer.

4. The method according to claim 1, wherein the oxidation solution does not contain the at least one stabilizer.

5. The method according to claim 1, wherein the oxidation solution contains a solvent selected from acetonitrile, toluene, dichloromethane, pyridine, N-methyl-2-pyrrolidone, and combinations thereof.

6. The method according to claim 1, wherein at least one stabilizer is sulfolane, and optionally, sulfolane may be the only stabilizer.

7. The method according to claim 1, wherein at least one stabilizer is TPP, and optionally, TPP may be the only stabilizer.

8. wherein at least one stabilizer is an uncrosslinked polymer, and the uncrosslinked polymer may be polystyrene, The method according to claim 1.

9. R is methyl, the sulfonyl oxidizing agent is methanesulfonyl azide (MsN₃), and the oxidation solution contains a solvent selected from acetonitrile, toluene, dichloromethane, pyridine, N-methyl-2-pyrrolidone, and combinations thereof, The method according to claim 1.

10. R is methyl, the sulfonyl oxidizing agent is methanesulfonyl azide (MsN₃), and the oxidation solution contains a solvent selected from acetonitrile, toluene, dichloromethane, pyridine, N-methyl-2-pyrrolidone, and combinations thereof, and at least one stabilizer is sulfolane or TPP, The method according to claim 1.

11. The oxidation solution is, containing 0.1 to 10 equivalents of the at least one stabilizer relative to the sulfonyl oxidizing agent, or, comprising from 1 to 10 equivalents of said at least one stabilizer relative to said sulfonyl oxidizing agent, or comprising from 3 to 6 equivalents of said at least one stabilizer relative to said sulfonyl oxidizing agent, or comprising from 4 to 5 equivalents of said at least one stabilizer relative to said sulfonyl oxidizing agent, The method according to any one of claims 1 to 10.

12. wherein the oxidation solution comprises from 0.1 to 10 M of said sulfonyl oxidizing agent, or comprises from 0.5 to 5 M of said sulfonyl oxidizing agent, or comprises from 0.6 to 1.5 M of said sulfonyl oxidizing agent, The method according to any one of claims 1 to 10.

13. A method for synthesizing a modified oligonucleotide comprising at least one internucleoside linkage of Formula I, 【Chemical 2】 wherein, for each internucleoside linkage of Formula I independently, X is selected from O and S, R is selected from aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, diazole, substituted diazole, C １ ~C ６ alkoxy, C １ ~C ２０ alkyl, C １ ~C ６ alkenyl, C １ ~C ６ alkynyl, substituted C １ ~C ２０ alkyl, substituted C １ ~C ６ alkenyl, substituted C １ ~C ６ alkynyl, and conjugate groups, said method comprising a) providing a solid support having a first blocked hydroxyl group attached thereto; b) adding to the reaction mixture a deblocking agent for deblocking said first blocked hydroxyl group so as to provide a first free hydroxyl group; c) adding to the reaction mixture a nucleoside so as to couple the nucleoside at said first free hydroxyl group so as to provide a triester phosphite linked nucleoside wherein said nucleoside comprises a phosphoramidite group and a second blocked hydroxyl group, said adding step; d) adding to the reaction mixture 1. a standard oxidizing agent for forming a phosphotriester internucleoside bond; 2. a standard sulfurizing agent for forming a phosphorothioate internucleoside bond, or 3. a sulfonyl oxidizing agent and at least one stabilizer for forming a sulfonyl phosphoramidate internucleoside bond, said adding step; wherein said stabilizer is selected from sulfolane, naphthalene, triphenyl phosphate (TPP), uncrosslinked polymer, polystyrene, soluble polymer, wax, triglyceride, and paraffin wax; e) optionally, treating said sulfonyl phosphoramidate bond, said phosphotriester bond, or said phosphorothioate bond with a capping reagent to cap any unreacted free hydroxyl groups. f) repeating steps b) to e) a predetermined number of times, with at least one iteration including step (d) 3, so as to provide the modified oligonucleotide; g) treating the modified oligonucleotide with triethylamine or diethylamine in acetonitrile; h) optionally, treating the modified oligonucleotide with ammonium hydroxide to cleave the modified oligonucleotide from the solid support; comprising; whereby the modified oligonucleotide comprising at least one internucleoside linkage of formula I is synthesized; the method. **Claim 14** The method according to claim 13, wherein each R is methyl. **Claim 15** The method according to claim 13, wherein the sulfonyl oxidizing agent is methanesulfonyl azide and the oxidizing solution comprises the methanesulfonyl azide and the at least one stabilizer. **Claim 16** The method according to claim 13, wherein the at least one stabilizer is sulfolane and, optionally, sulfolane may be the only stabilizer. **Claim 17** The method according to claim 13, wherein the at least one stabilizer is TPP and, optionally, TPP may be the only stabilizer. **Claim 18** R is methyl, and the sulfonyl oxidizing agent is methanesulfonyl azide and the oxidizing solution comprises the methanesulfonyl azide and the at least one stabilizer, The method according to claim 13. **Claim 19** R is methyl, and the sulfonyl oxidizing agent is methanesulfonyl azide and the oxidizing solution comprises the methanesulfonyl azide and the at least one stabilizer, and at least one stabilizer is sulfolane or TPP, The method according to claim 13. **Claim 20** The residue obtained by evaporation of the solvent from the solution containing the sulfonyl oxidizing agent and the at least one stabilizer has a combustion energy of less than 500 J.g －１ -1 or less than 300 J.g-1, The method according to any one of claims 1 to 10 or 13 to 19. **Claim 21** The method according to any one of claims 13 to 19, wherein each X is O. **Claim 22** The method according to any one of claims 13 to 19, wherein the capping reagent is acetic anhydride. **Claim 23** The method according to any one of claims 13 to 19, comprising treating the modified oligonucleotide with ammonium hydroxide to remove the protecting group and cleave the modified oligonucleotide from the solid support. **Claim 24** The method according to any one of claims 13 to 19, wherein the modified oligonucleotide comprises 12 to 25 linked nucleosides.

25. The method according to any one of claims 1 to 10 or 13 to 19, wherein the modified oligonucleotide may comprise phosphorothioate and mesylphosphoramidate, and optionally 7 bond(s) between phosphodiester nucleosides.

26. The method according to any one of claims 1 to 10 or 13 to 19, wherein the modified oligonucleotide comprises internucleoside bonds selected from phosphodiester, phosphorothioate, and mesylphosphoramidate internucleoside bond(s), and does not contain any other internucleoside bonds.

27. The method according to any one of claims 1 to 10 or 13 to 19, wherein the modified oligonucleotide comprises a stereospecific sugar moiety, cEt sugar moiety, 2'-MOE sugar moiety, 2'-OMe sugar moiety, 2'-F sugar moiety, 2'-NMA sugar moiety, and / or β-D-2'-deoxyribosyl sugar moiety.

28. The internucleoside bond of formula I or formula XIV is (a)adjacent to a nucleoside containing a cEt sugar moiety, 2'-MOE sugar moiety, 2'-OMe sugar moiety, 2'-F sugar moiety, 2'-NMA sugar moiety, and / or β-D-2'-deoxyribosyl sugar moiety, and / or (b)adjacent to a nucleoside containing an adenine, cytosine, 5-methylcytosine, guanine, thymine, or uracil nucleobase, The method according to any one of claims 1 to 10 or 13 to 19.

29. Further comprising attaching a conjugate group to form a conjugated modified oligonucleotide; Optionally, the conjugate group may comprise a cell targeting moiety. The method according to any one of claims 1 to 10 or 13 to 19.

30. The method according to claim 29, wherein the cell targeting moiety has an affinity for TfR.

31. The method according to claim 30, wherein the cell targeting moiety has an affinity for an asialoglycoprotein receptor (ASGPR).

32. A modified oligonucleotide synthesized by the method according to any one of claims 13 to 19, or an oligomeric compound comprising the modified oligonucleotide, or A modified oligonucleotide comprising a nucleoside internucleoside linkage synthesized by the method according to any one of claims 1 to 10, or an oligomeric compound comprising said modified oligonucleotide.

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