GLP-1 receptor ligand moiety conjugated oligonucleotides and uses thereof

Abstract

The present invention relates to GLP-1 receptor ligand moiety conjugated oligonucleotides and uses thereof. The present embodiments provide compounds and methods for targeting cells expressing the GLP-1 receptor.

Description

[0001] Sequence Listing

[0002] This application is submitted together with a sequence table in electronic format. The sequence table is provided as a file titled BIOL0320USL2SEQ_ST25.txt created on August 30, 2018, and its size is 29kb. The information of the sequence table in electronic format is incorporated herein by reference in its entirety. Technical Field

[0003] This example provides compounds and methods for targeting cells expressing the GLP-1 receptor. Background Art

[0004] The GLP-1 receptor is a class 2 G protein-coupled receptor that couples to adenylate cyclase via a stimulatory G protein receptor. Enterotrophic stimulation causes the release of glucagon-like peptide-1 into the circulation. Circulating GLP-1 binds to the GLP-1 receptor on pancreatic β islet cells. This activates the GLP-1 receptor, which induces signaling events that lead to insulin exocytosis of β islet cells. The binding between GLP-1 and the GLP-1 receptor causes the receptor to be internalized into the cytoplasm and eventually sorted into the lysosome (Kuna et al., 2013 Am J Physiol Endo Metab [American Journal of Physiology and Endocrinology Metabolism] 305: E161-E170). Summary of the invention

[0005] The embodiments provided herein are directed to compounds and methods for regulating the expression of nucleic acid targets in cells, wherein the cells express GLP-1 receptors. In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 receptor ligand conjugate portion. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 receptor ligand conjugate portion. In certain embodiments, cells expressing GLP-1 receptors (e.g., pancreatic β islet cells) are contacted with compounds provided herein to regulate the expression of nucleic acid targets in cells. In certain embodiments, compared to cells that do not express GLP-1 receptors, compounds comprising a GLP-1 receptor ligand conjugate portion selectively or preferentially target cells expressing GLP-1 receptors. In certain embodiments, compared to compounds that do not comprise a GLP-1 receptor ligand conjugate portion, compounds comprising a GLP-1 receptor ligand conjugate portion selectively or preferentially target cells expressing GLP-1 receptors. DETAILED DESCRIPTION

[0009] It should be understood that both the foregoing overview and the following detailed description are exemplary and illustrative only and do not limit the embodiments as required. In this article, the use of the singular includes the plural, unless otherwise expressly stated. As used herein, the use of "or" means "and / or", unless otherwise stated. In addition, the use of the term "including" and other forms (such as "includes" and "included") is not restrictive.

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

[0011] It should be understood that the sequence listed in each SEQ ID NO of oligonucleotide in the examples contained herein is independent of any modification to sugar moieties, internucleosides, or core bases.Thus, the oligonucleotide defined by SEQ ID NO can independently include one or more modifications to sugar moieties, internucleosides, or core bases.The oligonucleotide described by ISIS or ION numbering (ISIS# or ION#) indicates a combination of core base sequences, chemical modifications, and motifs.

[0012] It should be understood that, unless otherwise indicated, throughout the specification, the first letter in a peptide sequence is the first amino acid at the N-terminus of the peptide and the last letter in a peptide sequence is the last amino acid at the C-terminus of the peptide.

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

[0014] "2'-deoxynucleoside" means a nucleoside comprising a 2'-H(H)furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0015] "2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furanosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0016] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside comprising a 2'-MOE modified sugar moiety.

[0017] "2'-substituted nucleosides" or "2-modified nucleosides" means nucleosides comprising 2'-substituted or 2'-modified sugar moieties. As used herein, "2'-substituted" or "2-modified" with respect to sugar moieties means sugar moieties comprising at least one 2'-substituent group other than H or OH.

[0018] "5-methylcytosine" means cytosine with a methyl group attached to the 5 position.

[0019] "About" means within ±10% of a value. For example, if it is stated that "the compound affects about 70% inhibition of the target nucleic acid," it implies that the target nucleic acid level is inhibited within the range of 60% and 80%.

[0020] "Administration" or "administering" refers to the route by which a compound or composition provided herein is introduced into an individual's body to perform its intended function. Examples of routes of administration that can be used include, but are not limited to, parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

[0021] "Aminoisobutyric acid" or "Aib" means 2-aminoisobutyric acid having the formula:

[0022] Unless otherwise stated.

[0023] "Animal" refers to human or non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates (including but not limited to monkeys and chimpanzees).

[0024] "Antisense activity" means any detectable and / or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of the target nucleic acid or target protein level compared to the target nucleic acid level or the protein encoded by such a target nucleic acid in the absence of the antisense compound of the target.

[0025] "Antisense compound" means a compound comprising an oligonucleotide and optionally one or more additional features (e.g., conjugate groups or end groups). Examples of antisense compounds include single-stranded and double-stranded compounds, such as oligonucleotides, ribozymes, siRNA, shRNA, ssRNA, and occupancy-based compounds.

[0026] "Antisense inhibition" means a decrease in the level of a target nucleic acid in the presence of an antisense compound that is complementary to the target nucleic acid, compared to the level of the target nucleic acid in the absence of the antisense compound.

[0027] "Antisense mechanisms" are all those mechanisms involving hybridization of a compound to a target nucleic acid, wherein the result or effect of the hybridization is target degradation or target occupancy with concomitant shutdown of the cellular machinery involved, for example, in transcription or splicing.

[0028] "Antisense oligonucleotide" means an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid or a region or segment thereof. In certain embodiments, the antisense oligonucleotide can specifically hybridize to a target nucleic acid or a region or segment thereof.

[0029] "Bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety. "Bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

[0030] "Branch group" means a group of atoms having at least 3 positions capable of forming covalent bonds with at least 3 groups. In certain embodiments, the branch group provides multiple reactive sites for attaching a tethered ligand to an oligonucleotide via a conjugate linker and / or a cleavable moiety.

[0031] By "cell targeting moiety" is meant a conjugate group or a portion of a conjugate group that is capable of binding to a specific cell type or to specific cell types.

[0032] "cEt" or "constrained ethyl" means a bicyclic furanosyl sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'.

[0033] "Chemical modification" in a compound describes a substitution or alteration by a chemical reaction of any unit in the compound. "Modified nucleosides" means nucleosides independently having modified sugar moieties and / or modified nucleobases. "Modified oligonucleotides" means oligonucleotides comprising at least one modified internucleoside linkage, modified sugar, and / or modified nucleobase.

[0034] A "chemically distinct region" refers to a region of a compound that is chemically distinct to some extent from another region of the same compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without the 2'-O-methoxyethyl modification.

[0035] "Chimeric antisense compounds" refers to antisense compounds that have at least two chemically distinct regions, with multiple subunits at each position.

[0036] "Cleavable bond" means any chemical bond that can be separated. In certain embodiments, the cleavable bond is selected from: amide, polyamide, ester, ether, one or two esters of phosphodiester, phosphate, carbamate, disulfide, or peptide.

[0037] "Cleavable moiety" means a bond or group of atoms that is cleaved under physiological conditions, for example, within a cell, animal or human.

[0038] "Complementary" with respect to an oligonucleotide means that when the two nucleobase sequences are aligned in opposite directions, the nucleobase sequence of such an oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof. As described herein, nucleobase matching or complementary nucleobases are limited to the following base pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine ( m C) and guanine (G), unless otherwise indicated. Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at every nucleoside, and may include one or more nucleobase mismatches. In contrast, "fully complementary" or "100% complementary" with respect to oligonucleotides means that such oligonucleotides have nucleobase matches at every nucleoside without any nucleobase mismatches.

[0039] "Conjugate group" means a group of atoms attached to an oligonucleotide. A conjugate group includes a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[0040] "Conjugate linker" means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

[0041] "Conjugate moiety" means a group of atoms attached to an oligonucleotide via a conjugate linker.

[0042] "Design" or "designed" refers to the process of designing compounds that specifically hybridize to a selected nucleic acid molecule.

[0043] "Differently modified" means chemical modifications or chemical substituents that are different from each other, including the absence of modifications. Thus, for example, MOE nucleosides and unmodified DNA nucleosides are "differently modified", although the DNA nucleosides are unmodified. Similarly, DNA and RNA are "differently modified", even though both are naturally occurring unmodified nucleosides. Nucleosides that are identical except for containing different nucleobases are not differently modified. For example, a nucleoside containing a 2'-OMe modified sugar and an unmodified adenine nucleobase is not differently modified from a nucleoside containing a 2'-OMe modified sugar and an unmodified thymine nucleobase.

[0044] A "double-stranded antisense compound" means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two oligomeric compounds comprises an oligonucleotide.

[0045] "Expression" includes all functions by which the coding information of a gene is converted into structures that exist and operate in cells. Such structures include, but are not limited to, products of transcription and translation.

[0046] "Gapmer" means an oligonucleotide comprising an internal region having a plurality of nucleosides that supports RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the nucleosides or nucleosides comprising the external region. The internal region may be referred to as a "gap" and the external region may be referred to as a "wing".

[0047] "Hybridization" means the annealing of oligonucleotides and / or nucleic acids. Although not limited to a specific mechanism, the most common hybridization mechanisms involve hydrogen bonding that can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary nucleobases. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, antisense compounds and nucleic acid targets. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, oligonucleotides and nucleic acid targets.

[0048] "Inhibiting expression or activity" refers to a reduction or blocking of expression or activity relative to expression or activity in an untreated or control sample, and does not necessarily indicate complete elimination of expression or activity.

[0049] "Internucleoside linkage" means a group or bond that forms a covalent link between adjacent nucleosides in an oligonucleotide. "Modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring, phosphate internucleoside linkage. Non-phosphate linkages herein refer to modified internucleoside linkages.

[0050] "Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage.

[0051] "Linker-nucleosides" means nucleosides that connect an oligonucleotide to a conjugate portion. Linker-nucleosides are located within the conjugate linker of a compound. Linker-nucleosides are not considered to be part of the oligonucleotide portion of a compound (even if they are adjacent to an oligonucleotide).

[0052] "Mismatch" or "non-complementarity" means that when the first and second oligonucleotides are aligned, the nucleobase of the first oligonucleotide is not complementary to the corresponding nucleobase of the second oligonucleotide or the target nucleic acid. For example, a nucleobase (including but not limited to universal nucleobases, inosine, and hypoxanthine) is able to hybridize with at least one nucleobase, but is still mismatched or non-complementary with respect to the nucleobase to which it hybridizes. As another example, when the first and second oligonucleotides are aligned, a nucleobase of the first oligonucleotide that is not able to hybridize to the corresponding nucleobase of the second oligonucleotide or the target nucleic acid is a mismatch or non-complementary nucleobase.

[0053] "Regulate" refers to changing or adjusting a feature in a cell, tissue, organ, or organism. For example, regulating a target nucleic acid can mean raising or lowering the level of a target nucleic acid in a cell, tissue, organ, or organism. A "modulator" achieves the change in the cell, tissue, organ, or organism. For example, a compound can be a modulator that reduces the amount of a target nucleic acid in a cell, tissue, organ, or organism.

[0054] "MOE" means methoxyethyl.

[0055] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides.

[0056] "Motif" means the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages in an oligonucleotide.

[0057] "Natural" or "naturally occurring" means found in nature.

[0058] "Non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" means a modified sugar moiety that contains a modification (e.g., substitution) that does not form a bridge between two atoms of the sugar to form a second ring. "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

[0059] "Nucleobase" means a heterocyclic moiety capable of base pairing with another nucleic acid. As used herein, "naturally occurring nucleobases" are adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). "Modified nucleobases" are chemically modified naturally occurring nucleobases. "Universal bases" or "universal nucleobases" are nucleobases other than naturally occurring nucleobases and modified nucleobases, and are capable of pairing with any nucleobase.

[0060] "Nucleobase sequence" means the order of consecutive nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkages.

[0061] "Nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each independently unmodified or modified. "Modified nucleoside" means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides lacking a nucleobase.

[0062] By "oligomeric compound" is meant a compound comprising a single oligonucleotide and optionally one or more additional features (eg, conjugate groups or end groups).

[0063] "Oligonucleotide" means a polymer of connected nucleosides, each of which can be modified or unmodified independently of one another. Unless otherwise indicated, an oligonucleotide consists of 8-80 connected nucleosides. "Modified oligonucleotide" means an oligonucleotide in which at least one sugar, core base or internucleoside connection is modified. "Unmodified oligonucleotide" means an oligonucleotide that does not contain any sugar, core base or internucleoside modification.

[0064] "Parent oligonucleotide" means an oligonucleotide whose sequence is used as the basis for the design of more oligonucleotides having similar sequence but different length, motif, and / or chemistry. The newly designed oligonucleotide may have the same or overlapping sequence as the parent oligonucleotide.

[0065] "Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.

[0066] "Phosphorus moiety" means a group of atoms comprising a phosphorus atom. In certain embodiments, the phosphorus moiety comprises a mono-, di-, or tri-phosphate, or a thiophosphate.

[0067] "Portion" means a defined number of continuous (i.e., connected) core bases of a nucleic acid. In certain embodiments, a portion is a defined number of continuous core bases of a target nucleic acid. In certain embodiments, a portion is a defined number of continuous core bases of an oligomeric compound.

[0068] "Reduce" means to reduce the degree, size, amount or number.

[0069] "RNAi compound" means an antisense compound that acts at least in part through RISC or Ago2 rather than through RNase H to modulate 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).

[0070] A "segment" is defined as a smaller region or sub-portion of a region within a nucleic acid.

[0071] "Selectivity" with respect to an effect refers to a greater effect of one substance relative to another substance, by any quantitative degree or fold difference. For example, a compound comprising a GLP-1 receptor conjugate ligand portion is "selective" for cells expressing the GLP-1 receptor or "selectively targets" cells expressing the GLP-1 receptor, and the compound targets cells expressing the GLP-1 receptor to a greater extent than a compound that does not comprise a GLP-1 receptor conjugate ligand portion. As another example, a compound comprising a GLP-1 receptor conjugate ligand portion is "selective" for cells expressing the GLP-1 receptor or "selectively targets" cells expressing the GLP-1 receptor, and the compound targets cells expressing the GLP-1 receptor to a greater extent than cells that do not express or express relatively low levels of the GLP-1 receptor. It should be understood that the term "selectivity" does not require absolute all-or-nothing selectivity.

[0072] "Single-stranded" with respect to a compound means that the compound has only one oligonucleotide. "Self-complementary" means that an oligonucleotide hybridizes at least partially with itself. A compound consisting of one oligonucleotide, wherein the oligonucleotides of the compound are self-complementary, is a single-stranded compound. A single-stranded compound may be able to bind to a complementary compound to form a duplex.

[0073] A "site" is defined as a unique nucleobase position within a target nucleic acid.

[0074] "Specifically hybridizable to" means that an oligonucleotide has a sufficient degree of complementarity between the oligonucleotide and the target nucleic acid to induce the desired effect while exhibiting minimal or no effect on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.

[0075] "Specific inhibition" with respect to a target nucleic acid means that the expression of the target nucleic acid is reduced or blocked while exhibiting less, minimal, or no effect on non-target nucleic acids. Reduction does not necessarily indicate that the expression of the target nucleic acid is completely eliminated.

[0076] By "standard cellular assay" is meant one or more of the assays described in the Examples and reasonable variations thereof.

[0077] "Standard in vivo assays" means one or more of the procedures described in one or more of the Examples and reasonable variations thereof.

[0078] "Sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. "Unmodified sugar moiety" or "unmodified sugar" means a 2'-OH (H) furanose moiety as found in RNA ("unmodified RNA sugar moiety") or a 2'-H (H) moiety as found in DNA ("unmodified DNA sugar moiety"). Unmodified sugar moieties have one hydrogen at each of the 1', 3', and 4' positions, oxygen at the 3' position, and two hydrogens at the 5' position. "Modified sugar moiety" or "modified sugar" means a modified furanose sugar moiety or sugar surrogate. "Modified furanose sugar moiety" means a furanose sugar that includes a non-hydrogen substituent replacing at least one hydrogen of the unmodified sugar moiety. In certain embodiments, the modified furanose sugar moiety is a 2'-substituted sugar moiety. Such modified furanose sugar moieties include bicyclic sugars and non-bicyclic sugars.

[0079] "Sugar surrogate" means a sugar moiety having a modification other than a furanosyl moiety that can link a nucleobase to another group (e.g., an internucleoside linkage, a conjugate group, or a terminal group in an oligonucleotide). Modified nucleosides containing sugar surrogates can be incorporated into one or more positions within an oligonucleotide, and such oligonucleotides are capable of hybridizing to complementary compounds or nucleic acids.

[0080] "Target gene" refers to a gene encoding a target.

[0081] "Targeting" with respect to a target nucleic acid means specific hybridization of an oligonucleotide to said target nucleic acid to induce a desired effect. "Targeting" with respect to the GLP-1 receptor means binding of a GLP-1 receptor ligand conjugate moiety to the GLP-1 receptor.

[0082] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to a nucleic acid that can be targeted by the compounds described herein.

[0083] "Target region" means the portion of a target nucleic acid to which one or more compounds are targeted.

[0084] "Target segment" means the nucleotide sequence of the target nucleic acid to which the compound is targeted. "5' target site" refers to the 5'-most nucleotide of the target segment. "3' target site" refers to the 3'-most nucleotide of the target segment.

[0085] "Terminal group" means a chemical group or group of atoms covalently attached to the terminus of an oligonucleotide.

[0086] Certain embodiments

[0087] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 receptor ligand conjugate portion. In certain embodiments, the oligonucleotide is a modified oligonucleotide. In certain embodiments, the compound further comprises a conjugate linker. In certain embodiments, the conjugate linker connects the oligonucleotide to the GLP-1 receptor ligand conjugate portion.

[0088] In certain embodiments, the oligonucleotide is 8 to 80 linked nucleosides in length, 10 to 30 linked nucleosides in length, 12 to 30 linked nucleosides in length, or 15 to 30 linked nucleosides in length.

[0089] In certain embodiments, the oligonucleotide is a modified oligonucleotide comprising at least one modified internucleoside connection, at least one modified sugar or at least one modified core base. In certain embodiments, the modified internucleoside connection is a thiophosphate internucleoside connection. In certain embodiments, each modified internucleoside connection of the modified oligonucleotide is a thiophosphate internucleoside connection.

[0090] In certain embodiments, the modified sugar is a bicyclic sugar, such as 4'-(CH2)-O-2'(LNA); 4'-(CH2)2-O-2'(ENA); or 4'-CH(CH3)-O-2'(cEt). In certain embodiments, the modified sugar is 2'-O-methoxyethyl, 2'-F, or 2'-OMe.

[0091] In certain embodiments, the modified nucleobase is 5-methylcytosine.

[0092] In certain embodiments, the modified oligonucleotide comprises:

[0093] a gap segment consisting of linked deoxynucleosides;

[0094] a 5' wing segment consisting of linked nucleosides; and

[0095] a 3' wing segment consisting of linked nucleosides;

[0096] wherein the gap segment is positioned adjacent to and between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.

[0097] In certain embodiments, the oligonucleotide is single stranded.

[0098] In certain embodiments, the oligonucleotide is an antisense oligonucleotide, a miRNA antagonist, or a miRNA mimic.

[0099] In certain embodiments, the compound comprises a double-stranded duplex. In certain embodiments, the double-stranded duplex comprises a first strand and a second strand complementary to the first strand, wherein the first strand comprises a modified oligonucleotide. In certain embodiments, the first strand comprising a modified oligonucleotide is complementary to an RNA transcript. In certain embodiments, the second strand is complementary to an RNA transcript. In certain embodiments, the compound comprises a double-stranded duplex, wherein the duplex comprises (i) a first strand comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate portion, and (ii) a second strand complementary to the first strand. In certain embodiments, the compound comprises a double-stranded duplex, wherein the duplex comprises (i) a first strand comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate portion, and (ii) a second strand complementary to the first strand; wherein the first strand is complementary to an RNA transcript. In certain embodiments, the compound comprises a double-stranded duplex comprising (i) a first strand comprising a modified oligonucleotide, optionally a conjugate linker, and a GLP-1 receptor ligand conjugate moiety and (ii) a second strand complementary to the first strand; wherein the second strand is complementary to the RNA transcript.

[0100] In certain embodiments, the compound is a miRNA mimetic.

[0101] In certain embodiments, the compound comprises a ribonucleotide. In certain embodiments, the compound comprises a deoxyribonucleotide.

[0102] In certain embodiments, the oligonucleotide is complementary to an RNA transcript in a cell (eg, a pancreatic cell or a pancreatic β-islet cell).

[0103] In certain embodiments, the RNA transcript is a pre-mRNA, mRNA, non-coding RNA, or miRNA.

[0104] In certain embodiments, the GLP-1 receptor ligand conjugate moiety is a peptide conjugate moiety, a small molecule conjugate moiety, an aptamer conjugate moiety, or an antibody conjugate moiety that targets the GLP-1 receptor.

[0105] In certain embodiments, the peptide conjugate moiety is a GLP-1 peptide conjugate moiety.

[0106] In certain embodiments, the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0107] In certain embodiments, the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0108] In certain embodiments, the GLP-1 peptide conjugate portion is 8 to 50 amino acids in length and is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the amino acid sequence of any one of SEQ ID NOs: 1-57 over its entire length.

[0109] In certain embodiments, the entire length of the GLP-1 peptide conjugate portion is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0110] In certain embodiments, the GLP-1 peptide conjugate portion comprises at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 30, or 31 consecutive amino acid portions: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).

[0111] In certain embodiments, the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the same length portion of the amino acid sequence of GLP-1 (7-37).

[0112] In certain embodiments, the GLP-1 peptide conjugate portion is 8 to 50 amino acids in length and its entire length is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1).

[0113] In certain embodiments, the GLP-1 peptide conjugate moiety comprises conservative amino acid substitutions, amino acid analogs, or amino acid derivatives.

[0114] In certain embodiments, the entire length of the GLP-1 peptide conjugate portion is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of GLP-1(7-37) (SEQ ID NO:1).

[0115] In certain embodiments, the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0116] In certain embodiments, the GLP-1 peptide conjugate portion consists of the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0117] In certain embodiments, the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2 (SEQ ID NO: 2).

[0118] In certain embodiments, the GLP-1 peptide conjugate portion consists of the amino acid sequence of GLP-1(7-36)amide: the conventional three-letter code of the amino acid sequence is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2 (SEQ ID NO: 2).

[0119] In certain embodiments, the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence of GLP-1(7-36): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2).

[0120] In certain embodiments, the GLP-1 peptide conjugate portion comprises the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3).

[0121] In certain embodiments, the GLP-1 peptide conjugate portion consists of the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3).

[0122] In certain embodiments, the GLP-1 peptide conjugate portion comprises the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4)

[0123] In certain embodiments, the GLP-1 peptide conjugate portion consists of the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4).

[0124] In certain embodiments, the GLP-1 peptide conjugate portion comprises the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0125] In certain embodiments, the GLP-1 peptide conjugate portion consists of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0126] In certain embodiments, the GLP-1 peptide conjugate moiety may be the C-terminal amide or acid of any one of SEQ ID NOs: 1-57.

[0127] In certain embodiments, the GLP-1 peptide conjugate portion comprises the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Cys (SEQ ID NO: 22), wherein Aib is aminoisobutyric acid.

[0128] In certain embodiments, the GLP-1 peptide conjugate portion consists of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Cys (SEQ ID NO: 22), wherein Aib is aminoisobutyric acid.

[0129] In certain embodiments, the GLP-1 peptide conjugate portion comprises the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

[0130] In certain embodiments, the GLP-1 peptide conjugate portion consists of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

[0131] In certain embodiments, the GLP-1 peptide conjugate moiety is capable of binding to a GLP-1 receptor.

[0132] In certain embodiments, the GLP-1 receptor is expressed on the surface of a cell.

[0133] In certain embodiments, the cell is a pancreatic cell, such as a β-islet cell.

[0134] In certain embodiments, the cell is in an animal.

[0135] In certain embodiments, the compound comprises at least one, at least two, at least three, at least four, or at least five GLP-1 receptor ligand conjugate moieties.

[0136] In certain embodiments, the conjugate linker attaches the GLP-1 receptor ligand conjugate moiety to the 5' terminus of the oligonucleotide.

[0137] In certain embodiments, the conjugate linker attaches the GLP-1 receptor ligand conjugate moiety to the 3' terminus of the oligonucleotide.

[0138] In certain embodiments, the conjugate linker is cleavable.

[0139] In certain embodiments, the conjugate linker comprises a disulfide bond.

[0140] In certain embodiments, a disulfide bond links the GLP-1 peptide conjugate moiety to the oligonucleotide.

[0141] In certain embodiments, a disulfide bond connects the C-terminus of the GLP-1 peptide conjugate moiety to the 5' end of the oligonucleotide.

[0142] In certain embodiments, the conjugate linker comprises 1-5 linker-nucleosides.

[0143] In certain embodiments, the conjugate linker comprises 3 linker-nucleosides.

[0144] In certain embodiments, the three linker-nucleosides have a TCA motif.

[0145] In certain embodiments, 1-5 linker-nucleosides do not comprise a TCA motif.

[0146] In certain embodiments, the conjugate linker comprises a hexylamino group.

[0147] In certain embodiments, the conjugate linker comprises a polyethylene glycol group.

[0148] In certain embodiments, the conjugate linker comprises a triethylene glycol group.

[0149] In certain embodiments, the conjugate linker comprises a phosphate group.

[0150] In certain embodiments, the conjugate linker comprises:

[0151]

[0152] wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and

[0153] Y is directly or indirectly attached to the modified oligonucleotide. In certain embodiments, X comprises O. In certain embodiments, Y comprises a phosphate group. In certain embodiments, X is attached to the GLP-1 receptor ligand conjugate moiety via a disulfide bond.

[0154] In certain embodiments, the conjugate linker comprises:

[0155]

[0156] wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and

[0157] wherein T1 comprises the modified oligonucleotide; and Bx is a modified or unmodified nucleobase. In certain embodiments, X comprises a disulfide bond.

[0158] In certain embodiments, the conjugate linker comprises:

[0159]

[0160] in:

[0161] The phosphate group is linked to the modified oligonucleotide, and Y is linked to the conjugate group;

[0162] Y is a phosphodiester or amino (-NH-) group;

[0163] Z is a pyrrolidinyl group having the formula:

[0164]

[0165] j is 0 or 1;

[0166] n is from about 1 to about 10;

[0167] p is from 1 to about 10;

[0168] m is 0 or from 1 to 4; and

[0169] When Y is amino, m is 1.

[0170] In certain embodiments, Y is an amino group (-NH-) or a phosphodiester group. In certain embodiments, n is 3 and p is 3. In certain embodiments, n is 6 and p is 6. In certain embodiments, n is from 2 to 10 and p is from 2 to 10. In certain embodiments, n and p are different. In certain embodiments, n and p are the same. In certain embodiments, m is 0 or 1. In certain embodiments, j is 0. In certain embodiments, j is 1 and Z has the following formula:

[0171]

[0172] In certain embodiments, n is 2 and p is 3. In certain embodiments, n is 5 and p is 6.

[0173] In certain embodiments, the conjugate linker comprises:

[0174]

[0175] In certain embodiments, the conjugate linker comprises:

[0176]

[0177] In certain embodiments, the compound comprising a conjugate linker comprises:

[0178] in

[0179] NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; and

[0180] Y is attached directly or indirectly to the oligonucleotide.

[0181] In certain embodiments, the compound comprising a conjugate linker comprises:

[0182] in

[0183] NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; and

[0184] Y is attached directly or indirectly to the oligonucleotide.

[0185] In certain embodiments, the compound comprising a conjugate linker comprises:

[0186] in

[0187] NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; and

[0188] Y is attached directly or indirectly to the oligonucleotide.

[0189] In certain embodiments, compositions comprise at least one compound described herein. In certain embodiments, pharmaceutical compositions comprise at least one compound described herein and a pharmaceutically acceptable excipient.

[0190] In certain embodiments, the method for regulating the expression of a nucleic acid target in a cell comprises contacting the cell with a compound described in any one of the above embodiments, thereby regulating the expression of the nucleic acid target in the cell. In certain embodiments, the cell expresses a GLP-1 receptor on the cell surface. In certain embodiments, the cell is a pancreatic cell, such as a β-islet cell. In certain embodiments, the cell is a pituitary cell, a meningeal cell, a central nervous system (CNS) cell, a gastric cell, an intestinal cell, a duodenal cell, an ileal cell, a colon cell, a breast cell, a lung cell, a heart cell, a thyroid cell, or a kidney cell. In certain embodiments, the cell expressing the GLP-1 receptor on its surface is a cancer cell. In certain embodiments, cancer is an endocrine cancer, including but not limited to pheochromocytoma, paraganglioma, medullary thyroid carcinoma, adrenal cortical adenoma, parathyroid carcinoma, and pituitary adenoma. In certain embodiments, cancer is a nervous system cancer, including but not limited to meningioma, astrocytoma, glioblastoma, ependymoma, and schwannoma. In certain embodiments, the cancer is an embryonic carcinoma, including but not limited to medulloblastoma, Wilms tumor and neuroblastoma. In certain embodiments, the cancer includes but is not limited to ovarian cancer, prostate cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, lung cancer and lymphoma. In certain embodiments, the cell is contacted with the compound described in any one of the above embodiments to inhibit the expression of the nucleic acid target. In certain embodiments, the nucleic acid target is pre-mRNA, mRNA, non-coding RNA or miRNA. In certain embodiments, the cell is in an animal.

[0191] In certain embodiments, the method of regulating the expression of a nucleic acid target in an animal comprises administering to an animal a compound of any one of the above embodiments, thereby regulating the expression of a nucleic acid target in an animal. In certain embodiments, the expression of a nucleic acid target is regulated in an animal cell expressing a GLP-1 receptor on the cell surface. In certain embodiments, the expression of a nucleic acid target is regulated in a pancreatic cell of an animal, such as a β-islet cell. In certain embodiments, the cell is a pancreatic cell, such as a β-islet cell. In certain embodiments, the cell is a pituitary cell, a meningeal cell, a duodenal cell, an ileal cell, a colon cell, a breast cell, a lung cell, or a kidney cell. In certain embodiments, the cell expressing a GLP-1 receptor on its surface is a cancer cell. In certain embodiments, cancer is an endocrine cancer, including but not limited to pheochromocytoma, paraganglioma, medullary thyroid carcinoma, adrenocortical adenoma, parathyroid carcinoma, and pituitary adenoma. In certain embodiments, cancer is a nervous system cancer, including but not limited to meningioma, astrocytoma, glioblastoma, ependymoma, and schwannoma. In certain embodiments, the cancer is an embryonal carcinoma, including but not limited to medulloblastoma, Wilms tumor, and neuroblastoma. In certain embodiments, the cancer includes but is not limited to ovarian cancer, prostate cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, lung cancer, and lymphoma. In certain embodiments, the compound is administered to inhibit the expression of a nucleic acid target in an animal. In certain embodiments, the nucleic acid target is a pre-mRNA, mRNA, non-coding RNA, or miRNA.

[0192] Also provided herein is the use of a compound as described herein in the manufacture of a medicament for treating cancer. Also provided herein is the use of a compound as described herein for treating cancer.

[0193] In certain embodiments, the method of preparing a compound comprises:

[0194]

[0195] reacting with a GLP-1 peptide; wherein X1 is an oligonucleotide, and the compound is a GLP-1 peptide-conjugated oligonucleotide.

[0196] In certain embodiments, the method of preparing a compound comprises:

[0197] An oligonucleotide comprising a hexamethyl linker and a terminal amine is reacted at the 5' end of the oligonucleotide with 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) having the formula:

[0198]

[0199] Thus, compound 2 having the following formula is obtained:

[0200]

[0201] wherein X1 is an oligonucleotide; and

[0202] Compound 2 is reacted with a GLP-1 peptide to obtain a GLP-1 peptide-conjugated oligonucleotide having the following formula:

[0203]

[0204] wherein X1 is an oligonucleotide and X2 is a GLP-1 peptide.

[0205] In certain embodiments, the method of preparing a GLP-1 peptide-conjugated oligonucleotide comprises:

[0206] A solution containing an oligonucleotide comprising a hexamethyl linker and a terminal amine at the 5' end of the oligonucleotide is mixed with a solution containing 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) having the formula:

[0207]

[0208] Thus, compound 2 having the following formula is obtained:

[0209]

[0210] wherein X1 is an oligonucleotide; and

[0211] The solution containing Compound 2 is mixed with a solution containing the GLP-1 peptide, thereby obtaining a GLP-1 peptide-conjugated oligonucleotide having the following formula:

[0212]

[0213] wherein X1 is an oligonucleotide and X2 is a GLP-1 peptide.

[0214] In certain embodiments, the solution comprising the oligonucleotide comprises sodium phosphate buffer and the solution comprising 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) comprises dimethylformamide.

[0215] In certain embodiments, the solutions are mixed at room temperature.

[0216] In certain embodiments, the solution comprising Compound 2 further comprises acetonitrile and NaHCO 3 , and has a pH of about 8.0.

[0217] In certain embodiments, the solution comprising the GLP-1 peptide further comprises dimethylformamide.

[0218] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0219] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0220] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be 8 to 50 amino acids in length, and its entire length is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0221] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-57 over its entire length.

[0222] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , or 31 consecutive amino acids: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, the conventional three-letter code of the amino acid sequence is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).

[0223] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of GLP-1 (7-37).

[0224] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be 8 to 50 amino acids in length, and its entire length is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1).

[0225] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1) over its entire length.

[0226] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0227] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may consist of the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0228] In any of the above methods for preparing compounds or GLP-1 peptide-conjugated oligonucleotides, the GLP-1 peptide may comprise the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2 (SEQ ID NO: 2).

[0229] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may consist of the amino acid sequence of GLP-1 (7-36) amide (SEQ ID NO: 2).

[0230] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise the amino acid sequence of GLP-1(7-36): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2).

[0231] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3).

[0232] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be composed of the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3).

[0233] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG,Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4).

[0234] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be composed of the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG,Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4).

[0235] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise an amino acid sequence of any one of SEQ ID NOs: 1-57.

[0236] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may consist of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0237] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Cys (SEQ ID NO: 22), wherein Aib is aminoisobutyric acid.

[0238] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be composed of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Cys (SEQ ID NO: 22), wherein Aib is aminoisobutyric acid.

[0239] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

[0240] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can be composed of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

[0241] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise a reactive sulfur moiety.

[0242] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise penicillamine.

[0243] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the penicillamine may be linked to the C-terminus of the GLP-1 peptide.

[0244] Certain compounds comprising oligonucleotides

[0245] In certain embodiments, the compound described herein can be an antisense compound. In certain embodiments, the antisense compound comprises an oligomeric compound or consists of it. In certain embodiments, the oligomeric compound comprises an oligonucleotide, such as a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a core base sequence complementary to the core base sequence of the target nucleic acid.

[0246] In certain embodiments, the compounds described herein comprise or consist of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence that is complementary to a nucleobase sequence of a target nucleic acid.

[0247] In certain embodiments, compound or antisense compound is single-stranded.Such single-stranded compound or antisense compound comprises oligomeric compound or is composed of it.In certain embodiments, such oligomeric compound comprises oligonucleotide and optionally conjugate group, or is composed of oligonucleotide and optionally conjugate group.In certain embodiments, described oligonucleotide is antisense oligonucleotide.In certain embodiments, described oligonucleotide is modified.In certain embodiments, the oligonucleotide of single-stranded antisense compound or oligomeric compound comprises self-complementary core base sequence.

[0248] In certain embodiments, the compound is double-stranded. Such double-stranded compounds include oligonucleotides with a first modification in a region complementary to the target nucleic acid and oligonucleotides with a second modification in a region complementary to the first modified oligonucleotide. In certain embodiments, the modified oligonucleotide is an RNA oligonucleotide. In such embodiments, the thymine nucleobase in the modified oligonucleotide is replaced by a uracil nucleobase. In certain embodiments, the compound includes a conjugate group. In certain embodiments, one of the modified oligonucleotides is conjugated. In certain embodiments, both modified oligonucleotides are conjugated. In certain embodiments, the first modified oligonucleotide is conjugated. In certain embodiments, the second modified oligonucleotide is conjugated. In certain embodiments, the length of the first modified oligonucleotide is 12-30 connected nucleosides, and the length of the second modified oligonucleotide is 12-30 connected nucleosides. In certain embodiments, antisense compounds are double-stranded. Such double-stranded antisense compounds include a first oligomeric compound with a region complementary to the target nucleic acid and a second oligomeric compound with a region complementary to the first oligomeric compound. The first oligomeric compound of such double-stranded antisense compounds generally comprises a modified oligonucleotide and optionally a conjugate group, or is composed of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such double-stranded antisense compounds can be modified or unmodified. Any oligomeric compound or two oligomeric compounds of a double-stranded antisense compound can comprise a conjugate group. The oligomeric compound of a double-stranded antisense compound can include non-complementary protruding nucleosides.

[0249] In certain embodiments, the compound comprises a double-stranded duplex comprising (i) a first strand comprising a modified oligonucleotide, optionally a conjugate linker, and a GLP-1 receptor ligand conjugate moiety, and (ii) a second strand complementary to the first strand. In certain embodiments, the compound comprises a double-stranded duplex comprising (i) a first strand comprising a modified oligonucleotide, optionally a conjugate linker, and a GLP-1 receptor ligand conjugate moiety, and (ii) a second strand complementary to the first strand; wherein the first strand is complementary to an RNA transcript. In certain embodiments, the compound comprises a double-stranded duplex comprising (i) a first strand comprising a modified oligonucleotide, optionally a conjugate linker, and a GLP-1 receptor ligand conjugate moiety, and (ii) a second strand complementary to the first strand; wherein the second strand is complementary to an RNA transcript.

[0250] Examples of single-stranded and double-stranded compounds include, but are not limited to, oligonucleotides, siRNAs, microRNAs targeting oligonucleotides, and single-stranded RNAi compounds such as small hairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNA mimics.

[0251] In certain embodiments, the compounds described herein have a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a target segment of a targeted target nucleic acid.

[0252] In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 10 to 30. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 12 to 30. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 12 to 22. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 14 to 30. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 14 to 20. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 15 to 30. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 15 to 20. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 16 to 30. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 16 to 20. In certain embodiments, the compounds described herein comprise oligonucleotides of subunits connected in length from 17 to 30. In certain embodiments, the compounds described herein comprise oligonucleotides of 17 to 20 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides of 18 to 30 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides of 18 to 21 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides of 18 to 20 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides of 20 to 30 linked subunits in length. In other words, the length of such oligonucleotides is 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, the compounds described herein comprise oligonucleotides having a length of 14 linked subunits. In certain embodiments, the compounds described herein comprise oligonucleotides having a length of 16 linked subunits. In certain embodiments, the compounds described herein comprise oligonucleotides having a length of 17 linked subunits. In certain embodiments, the compounds described herein comprise oligonucleotides having a length of 18 linked subunits. In certain embodiments, the compounds described herein comprise oligonucleotides having a length of 19 linked subunits. In certain embodiments, the compounds described herein comprise an oligonucleotide that is 20 linked subunits in length.In other embodiments, the compounds described herein comprise an oligonucleotide of 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the compounds described herein comprise a length of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits, or a range defined by any two of the above values. In some embodiments, the linked subunits are nucleotides, nucleosides, or nucleobases.

[0253] In certain embodiments, the compound may further include other features or elements attached to the oligonucleotide, such as conjugate groups. In certain embodiments, such compounds are antisense compounds. In certain embodiments, such compounds are oligomeric compounds. In the embodiment where the conjugate group includes nucleosides (i.e., the conjugate group is connected to the nucleosides of the oligonucleotide), the nucleosides of the conjugate group are not counted in the length of the oligonucleotide.

[0254] In certain embodiments, the compound can be shortened or truncated. For example, a single subunit can be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation). A shortened or truncated compound targeting a nucleic acid can delete two subunits from the 5' end of the compound, or alternatively, two subunits can be deleted from the 3' end of the antisense compound. Alternatively, the deleted nucleosides can be dispersed throughout the compound.

[0255] When a single additional subunit is present in the elongated compound, the additional subunit may be located at the 5' or 3' end of the compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in a compound where two subunits are added to the 5' end of the compound (5' addition), or alternatively to the 3' end (3' addition). Alternatively, the added subunits may be dispersed throughout the compound.

[0256] It is possible to increase or decrease the length of a compound (e.g., an oligonucleotide) and / or introduce mismatched bases without eliminating activity (Woolf et al., (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:7305-7309, 1992; Gautschi et al., J. Natl. Cancer Inst [Journal of the National Cancer Institute of the United States]. 93:463-471, March 2001; Maher and Dolnick, Nuc. Acid. Res. [Nucleic Acids Research] 16:3341-3358, 1988). However, seemingly small changes in oligonucleotide sequence, chemistry, and motifs can result in large differences in one or more of the many properties required for clinical development (Seth et al., J. Med. Chem. [Journal of Medicinal Chemistry] 2009, 52, 10; Egli et al., J. Am. Chem. Soc. [Journal of the American Chemical Society] 2011, 133, 16642).

[0257] In certain embodiments, the compounds described herein are interfering RNA compounds (RNAi), including double-stranded RNA compounds (also referred to as short interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds act at least in part through the RISC pathway to degrade and / or chelate target nucleic acids (therefore, including microRNA / microRNA mimic compounds). As used herein, the term siRNA is intended to be equivalent to other terms used to describe nucleic acid molecules capable of mediating sequence-specific RNAi, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotides, short interfering nucleic acids, short interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is intended to be equivalent to other terms used to describe sequence-specific RNA interference (e.g., post-transcriptional gene silencing, translation inhibition, or epigenetics).

[0258] In certain embodiments, the first strand of the compound is a siRNA guide strand, and the second strand of the compound is a siRNA passenger strand. In certain embodiments, the second strand of the compound is complementary to the first strand. In certain embodiments, the length of each strand of the compound is 16, 17, 18, 19, 20, 21, 22, or 23 connected nucleosides. In certain embodiments, the first strand or the second strand of the compound may include a conjugate group.

[0259] In certain embodiments, the compounds described herein include modified oligonucleotides. Some modified oligonucleotides have one or more asymmetric centers and therefore produce enantiomers, diastereomers and other stereoisomeric configurations, and in terms of absolute stereochemistry, they can be defined as (R) or (S), or as α or β (for example, for sugar anomers), or as (D) or (L) (for example, for amino acids) etc. All such possible isomers are included in the modified oligonucleotides provided herein, including their racemic and optically pure forms, unless otherwise indicated. Similarly, all cis- and trans-isomers and tautomeric forms are also included.

[0260] The compounds described herein include variants in which one or more atoms are replaced with non-radioactive isotopes or radioactive isotopes of the specified elements. For example, a compound herein containing a hydrogen atom encompasses a 1 All possible deuterium substitutions of H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but are not limited to: 2 H or 3 H instead 1 H. 13 C or 14 C instead 12 C. 15 N instead 14 N. 17 O or 18 O instead 16 O. and 33 S. 34 S. 35 S or 36 S instead 32 S. In certain embodiments, non-radioactive isotope substitution can impart new properties to the compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotope substitution can render the compound suitable for research or diagnostic purposes, such as imaging assays.

[0261] Some mechanisms

[0262] In certain embodiments, the compounds described herein include or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds described herein include oligomeric compounds. In certain embodiments, the compounds described herein can hybridize to a target nucleic acid, thereby producing at least one antisense activity. In certain embodiments, the compounds described herein selectively affect one or more target nucleic acids. Such compounds include a core base sequence that hybridizes to one or more target nucleic acids, thereby producing one or more desired antisense activities, and do not hybridize to one or more non-target nucleic acids or do not hybridize to one or more non-target nucleic acids in such a way that produces a significant undesirable antisense activity.

[0263] In certain antisense activities, hybridization of the compounds described herein with target nucleic acids results in the recruitment of proteins that cut the target nucleic acids. For example, certain compounds described herein result in RNase H-mediated target nucleic acid cutting. RNase H is a cellular endonuclease that cuts the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex does not have to be unmodified DNA. In certain embodiments, the compounds described herein have enough "DNA-like" to cause RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleosides are tolerated in the gap of the gap body.

[0264] In certain antisense activities, a compound or a portion of a compound described herein is loaded into an RNA-induced silencing complex (RISC), which ultimately results in the cutting of a target nucleic acid. For example, certain compounds described herein result in the cutting of the target nucleic acid by Argonaute. The compound loaded into RISC is an RNAi compound. The RNAi compound can be double-stranded (siRNA) or single-stranded (ssRNA).

[0265] In certain embodiments, the hybridization of the compound described herein with the target nucleic acid does not result in the recruitment of proteins that cut the target nucleic acid. In certain such embodiments, the hybridization of the compound with the target nucleic acid results in a change in the splicing of the target nucleic acid. In certain embodiments, the hybridization of the compound with the target nucleic acid results in the inhibition of the binding interaction between the target nucleic acid and the protein or other nucleic acids. In certain such embodiments, the hybridization of the compound with the target nucleic acid results in a change in the translation of the target nucleic acid.

[0266] Antisense activity can be observed directly or indirectly. In certain embodiments, the observation or detection of antisense activity involves observing or detecting a change in the amount of a target nucleic acid or a protein encoded by such a target nucleic acid, a change in the ratio of a splice variant of a nucleic acid or protein, and / or a phenotypic change in a cell or animal.

[0267] Target nucleic acid, target region and nucleotide sequence

[0268] In certain embodiments, the compounds described herein comprise or consist of the following oligonucleotides, which comprise a region complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: mRNA and pre-mRNA, including intronic regions, exonic regions, and untranslated regions. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target nucleic acid is pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron / exon junction. In certain embodiments, at least 50% of the target region is within an intron. In certain embodiments, the target nucleic acid is in a cell expressing a GLP-1 receptor. In certain embodiments, the cell expressing the GLP-1 receptor is a pancreatic cell, such as a β-islet cell.

[0269] Hybridization

[0270] In some embodiments, hybridization occurs between a compound disclosed herein and a target nucleic acid.The most common hybridization mechanism involves hydrogen bonding between complementary nucleobases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding).

[0271] Hybridization can occur under different conditions. Hybridization conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

[0272] Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art.In certain embodiments, the compounds provided herein can specifically hybridize to a target nucleic acid.

[0273] Complementarity

[0274] When two core base sequences are compared in opposite directions, such an oligonucleotide or the core base sequence of one or more regions thereof matches the core base sequence of another oligonucleotide or nucleic acid or one or more regions thereof, and the oligonucleotide is said to be complementary to another nucleic acid. As described herein, core base matching or complementary core bases are limited to the following base pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G), unless otherwise stated. Complementary oligonucleotides and / or nucleic acids need not have core base complementarity at each nucleoside, and may include one or more core base mispairings. When such oligonucleotides have core base matching at each nucleoside without any core base mispairing, oligonucleotides are fully complementary or 100% complementary.

[0275] In certain embodiments, the compound described herein comprises or is composed of modified oligonucleotides. In certain embodiments, the compound described herein is an antisense compound. In certain embodiments, the compound comprises an oligomeric compound. The non-complementary core base between the compound and the target nucleic acid can be accepted, as long as the compound can still hybridize specifically with the target nucleic acid. In addition, the compound can be hybridized to one or more sections of the target nucleic acid so that interleaved or adjacent sections are not involved in hybridization events (for example, loop structure, mispairing or hairpin structure).

[0276] In certain embodiments, provided herein is a compound or its designated portion and, at least or at most with a target nucleic acid, its target region, target segment or designated portion is 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary. In certain embodiments, provided herein is a compound or its designated portion and a target nucleic acid, its target region, target segment or designated portion is 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100% or any number between these ranges complementary. The percentage complementarity of a compound and a target nucleic acid can be determined using conventional methods.

[0277] For example, 18 of the 20 core bases of the compound are complementary to the target region and therefore specifically hybridize to the compound representing 90% complementarity. In this example, the remaining non-complementary core bases can be clustered or interspersed with complementary core bases, and do not need to be continuous with each other or continuous with complementary core bases. Therefore, a compound with a length of 18 core bases and four non-complementary core bases flanked by two regions that are completely complementary to the target nucleic acid has an overall complementarity of 77.8% with the target nucleic acid. Using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J.Mol.Biol. [Molecular Biology], 1990, 215, 403410; Zhang and Madden, Genome Res. [Genome Research], 1997, 7, 649656) the complementarity percentage of the compound and the target nucleic acid region can be routinely determined. The percent homology, sequence identity or complementarity can be determined, for example, by the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482489) using the default settings.

[0278] In certain embodiments, the compounds described herein or their designated portions are fully complementary (i.e., 100% complementary) to the target nucleic acid or its designated portion. For example, the compound can be fully complementary to the target nucleic acid, or its target region or target segment or target sequence. As used herein, "fully complementary" means that each nucleobase of the compound is complementary to the corresponding nucleobase of the target nucleic acid. For example, a compound of 20 nucleobases is fully complementary to a target sequence of 400 nucleobases in length, as long as there are corresponding 20 nucleobase portions of the target nucleic acid that are fully complementary to the compound. Full complementarity can also be used with respect to the designated portion of the first and / or second nucleic acid. For example, the 20 nucleobase portions of a 30 nucleobase compound can be "fully complementary" to a target sequence of 400 nucleobases in length. The 20 nucleobase portions of a 30 nucleobase compound are fully complementary to the target sequence if the target sequence has corresponding 20 nucleobase portions, wherein each nucleobase is partially complementary to the 20 nucleobase portions of the compound. At the same time, the entire 30 nucleobase compound may or may not be completely complementary to the target sequence, depending on whether the remaining 10 nucleobases of the compound are also complementary to the target sequence.

[0279] In certain embodiments, relative to the target nucleic acid, the compound described herein comprises the core base of one or more mispairings.In certain such embodiments, the antisense activity for the target is reduced by such mispairing, but the activity for non-target is reduced by a larger amount.Therefore, in certain such embodiments, the selectivity of the compound is improved.In certain embodiments, the mispairing is specifically located in the oligonucleotide with a gap body motif.In certain such embodiments, the mispairing is at position 1, 2, 3, 4, 5, 6, 7 or 8 from the 5'-end of the gap region.In certain such embodiments, the mispairing is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3'-end of the gap region.In certain such embodiments, the mispairing is at position 1, 2, 3 or 4 from the 5'-end of the wing region.In certain such embodiments, the mispairing is at position 4, 3, 2 or 1 from the 3'-end of the wing region.In certain embodiments, the mispairing is specifically located in the oligonucleotide without a gap body motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5'-terminus of the oligonucleotide. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3'-terminus of the oligonucleotide.

[0280] The position of non-complementary core base can be at the 5' end or 3' end of described compound.Alternately, non-complementary core base or core base can be at the internal position of described compound.When there are two or more non-complementary core bases, they can be continuous (i.e. connected) or discontinuous.In one embodiment, non-complementary core base is located in the wing section of gap body oligonucleotide.

[0281] In certain embodiments, a compound described herein that is, or is up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprises no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase relative to a target nucleic acid (e.g., a target nucleic acid or a specified portion thereof).

[0282] In certain embodiments, a compound described herein that is, or is up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprises no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase relative to a target nucleic acid (e.g., a target nucleic acid or a specified portion thereof).

[0283] In certain embodiments, the compounds described herein also include those complementary to a portion of the target nucleic acid. As used herein, "portion" refers to a continuous (i.e., connected) core base of a determined number within a region or segment of the target nucleic acid. "Portion" may also refer to a continuous core base of a determined number of compounds. In certain embodiments, the compound is partially complementary to at least 8 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 9 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 10 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 11 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 12 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 13 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 14 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 15 core bases of the target segment. In certain embodiments, the compound is partially complementary to at least 16 core bases of the target segment. Also contemplated are compounds that are complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portions of a target segment, or a range defined by any two of these values.

[0284] Identity

[0285] Compounds provided herein may also have a determined percentage identity with a specific nucleotide sequence, SEQ ID NO, or a compound represented by a specific ISIS or ION number, or a portion thereof. In certain embodiments, compounds described herein are antisense compounds or oligomeric compounds. In certain embodiments, compounds described herein are modified oligonucleotides. As used herein, if a compound has the same nuclear base pairing ability as a sequence disclosed herein, the compound is identical to the sequence disclosed herein. For example, an RNA comprising uracil instead of the thymine in the disclosed DNA sequence is considered to be identical to the DNA sequence, because both uracil and thymine are paired with adenine. It is also contemplated that shortened and lengthened forms of compounds described herein and compounds with non-identical bases relative to compounds provided herein are present. Non-identical bases may be adjacent to each other or dispersed throughout the compound. The percentage identity of a compound is calculated according to the number of bases with the same base pairing relative to the sequence being compared with it.

[0286] In certain embodiments, the compounds described herein, or portions thereof, are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, at least to, or at most to, one or more of the compounds disclosed herein or SEQ ID NOs, or portions thereof. In certain embodiments, the compounds described herein are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, at least to, or at most to, one or more of the compounds disclosed herein or SEQ ID NOs, or portions thereof. In certain embodiments, the compounds described herein are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or any percentage between such values, wherein the compound comprises an oligonucleotide having one or more mismatched nucleobases. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5'-terminus of the oligonucleotide. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3'-terminus of the oligonucleotide.

[0287] In certain embodiments, the compounds described herein comprise or consist of antisense compounds. In certain embodiments, a portion of the antisense compounds is compared to the same length portion of the target nucleic acid. In certain embodiments, a portion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases is compared to the same length portion of the target nucleic acid.

[0288] In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide. In certain embodiments, a portion of the oligonucleotide is compared to the same length portion of the target nucleic acid. In certain embodiments, a portion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases is compared to the same length portion of the target nucleic acid.

[0289] Certain modified compounds

[0290] In certain embodiments, the compounds described herein include or consist of oligonucleotides, and the oligonucleotides are composed of connected nucleosides. The oligonucleotides can be unmodified oligonucleotides (RNA or DNA) or can be modified oligonucleotides. Relative to unmodified RNA or DNA, the modified oligonucleotides include at least one modification (i.e., including at least one modified nucleoside (comprising a modified sugar moiety and / or a modified core base) and / or at least one modified internucleoside connection).

[0291] A. Modified Nucleosides

[0292] Modified nucleosides contain a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

[0293] 1. Modified sugar moieties

[0294] In certain embodiments, the sugar moiety is a non-bicyclic modified sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogate may contain one or more substitutions corresponding to those of other types of modified sugar moieties.

[0295] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety comprising a furanosyl ring having one or more non-cyclic substituents, including but not limited to substituents at the 2', 4', and / or 5' positions. In certain embodiments, one or more non-cyclic substituents of the non-bicyclic modified sugar moiety are branched. Suitable At Examples of 2'-substituent groups of non-bicyclic modified sugar moieties include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2'-substituent group is selected from the group consisting of halogen, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C 10 Alkoxy, O-C1-C 10 Substituted alkoxy, O-C1-C 10 Alkyl, O-C1-C 10 Substituted alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )-alkynyl, O-alkylene-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ) or OCH2C(=O)-N(R m )(R n ), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10Alkyl, and 2'-substituent groups described in Cook et al., US6,531,584; Cook et al., US5,859,221; and Cook et al., US6,005,087. Certain embodiments of these 2'-substituent groups may be further substituted by one or more substituent groups independently selected from: hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4'-substituents suitable for linear non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015 / 106128. Examples of 5'-substituent groups suitable for non-bicyclic modified sugar moieties include, but are not limited to: 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugar comprises more than one non-bridging sugar substituent, such as a 2'-F-5'-methyl sugar moiety, and the modified sugar moieties and modified nucleosides described in Migawa et al. (WO 2008 / 101157) and Rajeev et al. (US 2013 / 0203836).

[0296] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent group selected from the group consisting of F, NH2, N3, OCF 3、 OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (OCH2C(=O)-N(R m )(R n )), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 alkyl.

[0297] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent group selected from the group consisting of 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").

[0298] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent group selected from the group consisting of F, OCH3, and OCH2CH2OCH3.

[0299] Nucleosides comprising modified sugar moieties (e.g., non-bicyclic modified sugar moieties) are referred to by the position of one or more substitutions on the sugar moiety of the nucleoside. For example, nucleosides comprising 2'-substituted or 2-modified sugar moieties are referred to as 2'-substituted nucleosides or 2-modified nucleosides.

[0300] The sugar moiety of some modifications comprises a bridging sugar substituent forming a second ring, thereby producing a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety comprises a bridge between 4' and 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (when in S configuration, referred to as "constrained ethyl" or "cEt ”), 4′-CH2-O-CH2-2′, 4′-CH2-N(R)-2′, 4′-CH(CH2OCH3)-O-2′ (“constrained MOE” or “cMOE”), and the like (see, e.g., Seth et al., U.S. Pat. No. 7,399,845; Bhat et al., U.S. Pat. No. 7,569,686; Swayze et al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 7,634,677; 8,022,193), 4'-C(CH3)(CH3)-O-2' and its analogs (see, e.g., Seth et al., US8,278,283), 4'-CH2-N(OCH3)-2' and its analogs (see, e.g., Prakash et al., US8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., Allerson et al., US7,696,345 and Allerson et al., US8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and its analogs (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, R a , and R b are independently H, a protecting group, or a C1-C 12 Alkyl (see, for example, Imanishi et al., US Pat. No. 7,427,672).

[0301] In certain embodiments, such 4' to 2' bridges independently comprise 1 to 4 linked groups independently selected from: -[C(R a )(R b )] n -、-[C(R a )(R b )] n -O-、-C(R a )=C(R b )-、-C(R a )=N-、-C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-、-S(=O) x -, and -N(R a )-;

[0302] in:

[0303] x is 0, 1, or 2;

[0304] n is 1, 2, 3, or 4;

[0305] Each R a and R b are independently H, a protecting group, a hydroxyl group, a C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl group (C(=O)-H), substituted acyl group, CN, sulfonyl group (S(=O)2-J1), or sulfoxyl group (sulfoxyl, S(=O)-J1); and each J1 and J2 are independently H, C1-C 12 Alkyl, substituted C1-C12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0306] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl.Acad.Sci.USA, 2000, 97, 5633-5638; Kumar et al., Bioorg.Med.Chem.Lett., 1998, 8, 2219-2222; Singh et al., J.Org.Chem., 1998, 63, 10035-10039; Srivastava et al., J.Am.Chem.Soc., 20017, 129, 8362-8379; Elayadi et al., Curr.Opinion Invens. Drugs [Investigational Drugs], 2001, 2, 558-561; Braasch et al., Chem. Biol. [Chemical Biology], 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther. [Current Opinion in Molecular Therapy], 2001, 3, 239-243; Wengel et al., US7,053,207, Imanishi et al., US6,268,490, Imanishi et al., US6,770,748, Imanishi et al., USRE44,779; Wengel et al., US6,794,499, Wengel et al., US6,670,461; Wengel et al., US7,034,133, Wengel et al., US8,080,644; Wengel et al., US8,034,909; Wengel et al., US8,153,365; Wengel et al., US7,572,582; and Ramasamy et al., US6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO 91999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., US 7,547,684; Seth et al., US7,666,854; Seth et al., US8,088,746; Seth et al., US7,750,131; Seth et al., US8,030,467; Seth et al., US8,268,980; Seth et al., US8,546,556; Seth et al., US8,530,640; Migawa et al., US9,012,421; Seth et al., US8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008 / 0039618 and Migawa et al., US2015 / 0191727. .

[0307] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.For example, LNA nucleosides (described herein) can be in the α-L configuration or in the β-D configuration.

[0308]

[0309] α-L-methyleneoxy (4'-CH2-O-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, the general description of bicyclic nucleosides includes two isomeric configurations. When the position of a specific bicyclic nucleoside (e.g., LNA or cEt) is identified in the exemplary embodiments herein, they are in the β-D configuration unless otherwise stated.

[0310] In certain embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (eg, 5'-substituted and 4'-2' bridged sugars).

[0311] In certain embodiments, the sugar moiety of modification is a sugar substitute. In certain such embodiments, the oxygen atom of the sugar moiety is replaced by, for example, sulfur, carbon or nitrogen atom. In certain such embodiments, the sugar moiety of such modification also includes bridging and / or non-bridging substituents as described herein. For example, some sugar substitutes include 4'-sulfur atoms and substitutions at 2'-positions (see, for example, Bhat et al., US7,875,733 and Bhat et al., US7,939,677) and / or 5' positions.

[0312] In certain embodiments, the sugar substitute comprises a ring having atoms other than 5. For example, in certain embodiments, the sugar substitute 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 acids ("HNA"), anitol nucleic acids ("ANA"), mannitol nucleic acids ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. [Bioorganic Chemistry and Medicinal Chemistry] 2002, 10, 841-854), fluoro HNA:

[0313]

[0314] ("F-HNA", see, e.g., Swayze et al., US8,088,904; Swayze et al., US8,440,803; Swayze et al., US; and Swayze et al., US9,005,906, F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:

[0315]

[0316] Wherein, independently, for each of the modified THP nucleosides:

[0317] Bx is the nucleobase portion;

[0318] T3 and T4 are each independently an internucleoside linking group that connects the modified THP nucleoside to the rest of the oligonucleotide, or one of T3 and T4 is an internucleoside linking group that connects the modified THP nucleoside to the rest of the oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and R1 and R2 are each independently selected from: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 are independently H or C1-C6 alkyl.

[0319] In certain embodiments, modified THP nucleosides are provided, wherein each of Q1, Q2, Q3, Q4, Q5, Q6 and Q7 is H. In certain embodiments, at least one of Q1, Q2, Q3, Q4, Q5, Q6 and Q7 is different from H. In certain embodiments, at least one of Q1, Q2, Q3, Q4, Q5, Q6 and Q7 is methyl. In certain embodiments, modified THP nucleosides are provided, wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.

[0320] In certain embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholinyl sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry [biochemistry], 2002, 41, 4503-4510 and Summerton et al., US5,698,685; Summerton et al., US5,166,315; Summerton et al., US5,185,444; and Summerton et al., US5,034,506). As used herein, the term "morpholinyl" means a sugar substitute having the following structure:

[0321]

[0322] In certain embodiments, the morpholinyl group can be modified, for example, by adding or changing different substituent groups from the above morpholinyl structure. Such sugar surrogates are referred to herein as "modified morpholinyl groups".

[0323] In certain embodiments, the sugar surrogate comprises a non-cyclic portion. Examples of nucleosides and oligonucleotides comprising such non-cyclic sugar surrogates include, but are not limited to, peptide nucleic acids ("PNAs"), non-cyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and Manoharan et al., nucleosides and oligonucleotides described in WO 2011 / 133876.

[0324] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

[0325] 2. Modified Nucleobases

[0326] Nucleobase (or base) modifications or substitutions are structurally distinguishable from naturally occurring or synthetic unmodified nucleobases, but functionally interchangeable therewith. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may confer nuclease stability, binding affinity, or some other beneficial biological property to antisense compounds.

[0327] In certain embodiments, the compounds described herein include modified oligonucleotides. In certain embodiments, the modified oligonucleotides include one or more nucleosides comprising unmodified nucleobases. In certain embodiments, the modified oligonucleotides include one or more nucleosides comprising modified nucleobases. In certain embodiments, the modified oligonucleotides include one or more nucleosides not comprising nucleobases (referred to as abasic nucleosides).

[0328] In certain embodiments, the modified nucleobase is selected from: 5-substituted pyrimidine, 6-azapyrimidine, alkyl or alkynyl substituted pyrimidine, alkyl substituted purine, and N-2, N-6 and O-6 substituted purine. In certain embodiments, the modified nucleobase is selected from: 2-aminopropyladenine, 5-hydroxymethylcytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (C≡C-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6 -azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted purines, 5-halo, (especially 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine Purines, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases Bases. Additional modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenthiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in Merigan et al., US Pat. No. 3,687,808, those disclosed in 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 those disclosed in Antisense Drug Technology, Chapters 6 and 15, Crooke ST, ed., CRC Press, 2008, 163-166 and 442-443.

[0329] Publications that teach the preparation of some of the modified nucleobases noted above, as well as other modified nucleobases, include, but are not limited to, Manoharan et al., US 2003 / 0158403; Manoharan et al., US 2003 / 0175906; Dinh et al., 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., US5,484,908; Matteucci et al., US5,502,177; Hawkins et al., US5,525,711; Haralambidis et al., US5,552,540; Cook et al., US5 ,587,469; Froehler et al., US5,594,121; Switzer et al., US5,596,091; Cook et al., US5,614,617; Froehler et al., US5,645,985; Cook et al., US5,681,941; Cook et al., US5,811,534; Cook et al., US5,750,692; Cook et al., US5,948,903; Cook et al., US5,587,470; Cook et al., US5,457,191; Matteucci et al., US5,763,588; Froehler et al., US5,830,653; Cook et al., US5,808,027; Cook et al., US6,166,199; and Matteucci et al., US6,005,096.

[0330] In certain embodiments, the compound of the target nucleic acid comprises one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

[0331] 3. Modified internucleoside linkages

[0332] The naturally occurring internucleoside linkages of RNA and DNA are 3' to 5' phosphodiester linkages. In certain embodiments, compounds described herein having one or more modified (i.e., non-naturally occurring) internucleoside linkages are selected over compounds having naturally occurring internucleoside linkages due to desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0333] In certain embodiments, the compound of the target nucleic acid comprises one or more modified internucleoside connections. In certain embodiments, the modified internucleoside connection is a phosphorothioate connection. In certain embodiments, each internucleoside connection of antisense compounds is a phosphorothioate internucleoside connection.

[0334] In certain embodiments, the compound described herein comprises an oligonucleotide. The oligonucleotide with modified internucleoside connections includes an internucleoside connection retaining a phosphorus atom and an internucleoside connection without a phosphorus atom. Representational internucleoside connections containing phosphorus include but are not limited to phosphodiester, phosphotriester, methylphosphonate, phosphoramidate and thiophosphate. The method for preparing phosphorus-containing connections and non-phosphorus-containing connections is well known.

[0335] In certain embodiments, any internucleoside linkage can be used to link the nucleosides of the modified oligonucleotide together. Two major types of internucleoside linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates (also referred to as unmodified or naturally occurring linkages) containing phosphodiester bonds ("P=O"), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S") and phosphorodithioates ("HS-P=S"). Representative non-phosphorus-containing internucleoside linkage groups include, but are not limited to, methylene azomethine (-CH2-N(CH3)-O-CH2-), thiodiesters, thiocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to naturally occurring phosphate linkages, modified internucleoside linkages can be used to change (typically increase) the nuclease resistance of oligonucleotides. In certain embodiments, the nucleoside with chiral atoms can be prepared as a racemic mixture, or prepared as a single enantiomer. Representational chiral nucleoside connections include but are not limited to alkyl phosphonates and thiophosphates. The method for preparing phosphorus-containing nucleoside connections and non-phosphorus-containing nucleoside connections is well known to those skilled in the art.

[0336] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Additional neutral internucleoside linkages include nonionic linkages comprising siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and P.D. Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH2 moieties.

[0337] In certain embodiments, oligonucleotide comprises the internucleoside connection of the modification of the internucleoside connection motif that is arranged into the restriction pattern or modification along described oligonucleotide or its region.In certain embodiments, internucleoside connection is arranged into the motif with gap.In such embodiments, the internucleoside connection in each of two wing regions is different from the internucleoside connection in the gap region.In certain embodiments, the internucleoside connection in wing is phosphodiester, and the internucleoside connection in the gap is thiophosphate.Select nucleoside motif independently, so this type of oligonucleotide with the internucleoside connection motif with gap can have or can not have the nucleoside motif with gap, and if it does have the nucleoside motif with gap, then wing and gap length can be identical or can be different.

[0338] In certain embodiments, the oligonucleotide comprises a region with an alternating internucleoside connection motif. In certain embodiments, the oligonucleotide comprises a region of unanimously modified internucleoside connections. In certain such embodiments, the oligonucleotide comprises a region uniformly connected by thiophosphate internucleoside connections. In certain embodiments, the oligonucleotide is uniformly connected by thiophosphate. In certain embodiments, each internucleoside connection of the oligonucleotide is selected from phosphodiester and thiophosphate. In certain embodiments, each internucleoside connection of the oligonucleotide selects phosphodiester and thiophosphate, and at least one internucleoside connection is thiophosphate.

[0339] In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside connections. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside connections. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside connections. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 continuous phosphorothioate internucleoside connections. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 continuous phosphorothioate internucleoside connections. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 continuous phosphorothioate internucleoside connections. In certain embodiments, the oligonucleotide comprises at least one block of at least 12 continuous phosphorothioate internucleoside connections. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides at the 3' end of the oligonucleotide.

[0340] In certain embodiments, oligonucleotides include one or more methylphosphonate connections. In certain embodiments, oligonucleotides with gap body nucleoside motifs include connection motifs that include all thiophosphate connections except one or two methylphosphonate connections. In certain embodiments, a methylphosphonate is connected in the central gap of an oligonucleotide with a gap body nucleoside motif.

[0341] In certain embodiments, it is desirable to arrange the number of connections between thiophosphate nucleosides and phosphodiester nucleosides to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of connections between thiophosphate nucleosides and the number and position of connections between phosphodiester nucleosides to maintain nuclease resistance. In certain embodiments, the number of connections between thiophosphate nucleosides can be reduced, and the number of connections between phosphodiester nucleosides can be increased. In certain embodiments, the number of connections between thiophosphate nucleosides can be reduced, and the number of connections between phosphodiester nucleosides can be increased, while still maintaining nuclease resistance. In certain embodiments, it is desirable to reduce the number of connections between thiophosphate nucleosides, while retaining nuclease resistance. In certain embodiments, it is desirable to increase the number of connections between phosphodiester nucleosides, while retaining nuclease resistance.

[0342] 4. Certain motifs

[0343] In certain embodiments, the compounds described herein include oligonucleotides. Oligonucleotides can have motifs, such as unmodified and / or modified sugar moieties, core bases, and / or internucleoside connections. In certain embodiments, the modified oligonucleotides include one or more modified nucleosides, and the nucleosides include modified sugars. In certain embodiments, the modified oligonucleotides include one or more modified nucleosides, and the nucleosides include modified core bases. In certain embodiments, the modified oligonucleotides include one or more modified internucleosides. In such embodiments, the modified, unmodified, and differently modified sugar moieties, core bases, and / or internucleosides of the modified oligonucleotides are connected to define patterns or motifs. In certain embodiments, the patterns of sugar moieties, core bases, and internucleosides are each independent of each other. Therefore, the modified oligonucleotides can be described by their sugar motifs, core base motifs, and / or internucleoside connection motifs (as used herein, independent of the sequence of the core base, the core base motif describes the modification of the core base).

[0344] 1. Certain sugar motifs

[0345] In certain embodiments, the compounds described herein include oligonucleotides. In certain embodiments, the oligonucleotides include one or more types of modified sugars and / or unmodified sugar moieties arranged into a defined pattern or sugar motif along the oligonucleotide or its region. In some cases, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.

[0346] In certain embodiments, the oligonucleotide of modification comprises or is composed of a region with a gap body motif, and the gap body motif comprises two external regions or "wings" and a center or inner region or "gap". The three regions of the gap body motif (5'-wing, gap, and 3'-wing) form the nucleoside of a continuous sequence, wherein at least some of the sugar moieties of each nucleoside in the wing are different from at least some of the sugar moieties of the nucleoside of the gap. Specifically, the sugar moieties nearest to the gap of the nucleoside of at least each wing (the most 3' nucleosides of the 5'-wing and the most 5' nucleosides of the 3'-wing) are different from the sugar moieties of adjacent gap nucleosides, therefore defining a boundary (that is, wing / gap junction) between the wing and the gap. In certain embodiments, the sugar moieties in the gap are identical to each other. In certain embodiments, the gap includes one or more nucleosides with the following sugar moieties, and the sugar moieties are different from the sugar moieties of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of these two wings are identical to each other (symmetric gap bodies). In certain embodiments, the sugar motifs of the 5'-wing are different from the sugar motifs of the 3'-wing (asymmetric gap bodies).

[0347] In certain embodiments, the wings of the gap body contain 1-5 nucleosides. In certain embodiments, the wings of the gap body contain 2-5 nucleosides. In certain embodiments, the wings of the gap body contain 3-5 nucleosides. In certain embodiments, the nucleosides of the gap body are all modified nucleosides.

[0348] In certain embodiments, the gap of the gap body comprises 7-12 nucleosides. In certain embodiments, the gap of the gap body comprises 7-10 nucleosides. In certain embodiments, the gap of the gap body comprises 8-10 nucleosides. In certain embodiments, the gap of the gap body comprises 10 nucleosides. In a certain embodiment, each nucleoside of the gap of the gap body is an unmodified 2'-deoxynucleoside.

[0349] In certain embodiments, the gap body is a deoxy gap body. In such embodiments, the nucleoside on the gap side of each wing / gap junction is an unmodified 2'-deoxynucleoside, and the nucleoside on the wing side of each wing / gap junction is a modified nucleoside. In some such embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In some such embodiments, each nucleoside of each wing is a modified nucleoside.

[0350] In certain embodiments, the oligonucleotide of modification has a sugar motif modified completely, wherein each nucleoside of the oligonucleotide modified comprises a sugar moiety modified. In certain embodiments, the oligonucleotide of modification comprises a region with a sugar motif modified completely, or consists of, wherein each nucleoside in the region comprises a sugar motif modified. In certain embodiments, the oligonucleotide of modification comprises a region with a sugar motif modified completely or consists of, wherein each nucleoside in the region modified completely comprises an identical sugar motif modified, referred to herein as an identically modified sugar motif. In certain embodiments, the oligonucleotide of modification completely is an identically modified oligonucleotide. In certain embodiments, each nucleoside of an identically modified oligonucleotide comprises an identical 2'-modification.

[0351] 2. Certain nucleobase motifs

[0352] In certain embodiments, the compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise modified and / or unmodified nucleobases arranged into a defined pattern or motif along the oligonucleotide or its region. In certain embodiments, each nucleobase is modified. In certain embodiments, the nucleobases are unmodified. 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 oligonucleotides are 5-methylcytosine.

[0353] In certain embodiments, the modified oligonucleotide comprises a block of modified nucleobases. In certain such embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides at the 3'-end of the oligonucleotide. In certain embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides at the 5'-end of the oligonucleotide.

[0354] In certain embodiments, the oligonucleotide with the gap body motif comprises a nucleoside comprising a modified nucleobase. In certain such embodiments, a nucleoside comprising a modified nucleobase is in the central gap of the oligonucleotide with the gap body motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2'-deoxyribose moiety. In certain embodiments, the modified nucleobase is selected from: 2-thiopyrimidine and 5-propyne pyrimidine.

[0355] 3. Certain internucleoside linkage motifs

[0356] In certain embodiments, the compounds described herein include oligonucleotides. In certain embodiments, oligonucleotides include modified and / or unmodified internucleoside connections arranged into a defined pattern or motif along the oligonucleotide or its region. In certain embodiments, substantially each internucleoside linking group is a phosphate internucleoside connection (P=O). In certain embodiments, each internucleoside linking group of the modified oligonucleotide is a thiophosphate (P=S). In certain embodiments, each internucleoside linking group of the modified oligonucleotide is independently selected from thiophosphate and phosphate internucleoside connections. In certain embodiments, the sugar motif of the modified oligonucleotide is a gap body, and the internucleoside connections in the gap are all modified. In certain such embodiments, some or all of the internucleoside connections in the wing are unmodified phosphate connections. In certain embodiments, the terminal internucleoside connection is modified.

[0357] 5. Certain modified oligonucleotides

[0358] In certain embodiments, the compound described herein comprises the oligonucleotide of modification.In certain embodiments, the above modification (sugar, core base, internucleoside connection) is incorporated into the oligonucleotide of modification.In certain embodiments, the oligonucleotide of modification is characterized by its modification, motif and total length.In certain embodiments, such parameters are each independent of one another.Therefore, unless otherwise indicated, each internucleoside connection of the oligonucleotide with gap body sugar motif can be modified or unmodified, and can follow or may not follow the gap body modification pattern of sugar modification.For example, the internucleoside connection in the wing region of sugar gap body can be the same or different from each other, and can be the same or different from the internucleoside connection in the gap region of sugar motif.Similarly, independent of such sugar-modified gap body pattern, such gap body oligonucleotide can include one or more modified core bases.In addition, in some cases, oligonucleotide is described by total length or range and by the length or length range of two or more regions (for example, the region with specified sugar-modified nucleoside), in such cases, the numerical value of each scope for the oligonucleotide with total length exceeding the specified range can be selected.In such cases, two factors must be satisfied. For example, in certain embodiments, the oligonucleotide of modification is made up of the nucleoside of 15-20 connections and has the sugar motif composed of three regions (A, B and C), wherein region A is made up of the nucleoside of 2-6 connections with specified sugar motif, region B is made up of the nucleoside of 6-10 connections with specified sugar motif, and region C is made up of the nucleoside of 2-6 connections with specified sugar motif.Such embodiment does not include the oligonucleotide of modification, wherein A and C are each made up of the nucleoside of 6 connections, and B is made up of the nucleoside of 10 connections (even if those quantities of nucleoside are allowed in the requirement of A, B and C), because the total length of such oligonucleotide is 22, the length exceeds the upper limit (20) of the total length of the oligonucleotide of modification.Herein, if the description relative to one or more parameter oligonucleotide is unmentioned, then such parameter is not limited.Therefore, the oligonucleotide of modification that is only described as having gap body sugar motif and not further described can have any length, internucleoside connection motif and core base motif.Unless otherwise indicated, all modifications are independent of core base sequence.

[0359] Certain conjugated compounds

[0360] In certain embodiments, the compounds described herein include oligonucleotides (modified or unmodified) and optionally one or more conjugate groups and / or end groups or are composed of them. The conjugate group is composed of one or more conjugate moieties and conjugate joints, and the conjugate joints connect the conjugate moieties to the oligonucleotides. The conjugate group can be attached to either end or both ends of the oligonucleotide and / or attached 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 end or both ends of the oligonucleotide is an end group. In certain such embodiments, the conjugate group or end group is attached to the 3' and / or 5'-end of the oligonucleotide. In certain such embodiments, the conjugate group (or end group) is attached to the 3'-end of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 3'-end of the oligonucleotide. In certain embodiments, the conjugate group (or end group) is attached to the 5'-end of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 5 '-terminus of the oligonucleotide.

[0361] Examples of terminal groups include, but are not limited to, conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more independently modified or unmodified nucleosides. GLP-1 Receptor Ligand Conjugate Moieties

[0362] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 receptor ligand conjugate portion. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 receptor ligand conjugate portion. In certain embodiments, the conjugate linker connects the GLP-1 receptor ligand conjugate portion to the oligonucleotide. In certain embodiments, the oligonucleotide is a modified oligonucleotide. In certain embodiments, the GLP-1 receptor ligand conjugate portion comprises a small molecule, an aptamer, an antibody, or a peptide.

[0363] 1. Certain GLP-1 receptor small molecule conjugates

[0364] In certain embodiments, the compound comprises an oligonucleotide and a small molecule conjugate portion capable of binding to a GLP-1 receptor. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a small molecule conjugate portion capable of binding to a GLP-1 receptor. In certain embodiments, the oligonucleotide is a modified oligonucleotide.

[0365] Any small molecule conjugate moiety capable of binding to the GLP-1 receptor known in the art can be used in several embodiments. For example, in certain embodiments, the small molecule conjugate moiety capable of binding to the GLP-1 receptor is a small molecule GLP-1 receptor antagonist as described in Willard et al., "Small Molecule Drug Discovery at the Glucagon-like Peptide-1 Receptor," Experimental Diabetes Research, vol. 2012, pp. 1-9; Sloop et al., "Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets," Diabetes, vol. 59, 2010, pp. 3099-3107; Knudsen et al., "Small-molecule agonists for the glucagon-like peptide1receptor," [Small molecule agonists of glucagon-like peptide-1 receptor] PNAS [Proceedings of the National Academy of Sciences of the United States of America] January 16, 2007; 104(3):937-42; or Wang et al., "Non-peptidic glucose-like peptide-1 receptor agonists: after math of a serendipitous discovery," [Non-peptidic glucose-like peptide-1 receptor agonists: after math of a serendipitous discovery] Acta Pharmacologica Sinica [Chinese Pharmacological Journal] (2010) 31:1026-1030; which are incorporated herein by reference in their entirety.

[0366] In certain embodiments, the small molecule conjugate moiety capable of binding to the GLP-1 receptor has any one of the following formulae:

[0367]

[0368]

[0369] 2. Certain GLP-1 receptor antibody conjugate moieties

[0370] In certain embodiments, the compound comprises an oligonucleotide and an antibody or fragment thereof that can bind to a GLP-1 receptor. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and an antibody or fragment thereof that can bind to a GLP-1 receptor. In certain embodiments, the oligonucleotide is a modified oligonucleotide. Any antibody or fragment thereof that can bind to a GLP-1 receptor known in the art can be used in several embodiments. In certain embodiments, the compound comprises an oligonucleotide and an antibody or fragment thereof that can bind to a GLP-1 receptor described in WO 2005018536, US20060275288, US 8,389,689, or WO 2011056644, which is incorporated herein by reference in its entirety. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and an antibody or fragment thereof capable of binding to the GLP-1 receptor described in WO 2005018536, US 20060275288, US 8,389,689, or WO 2011056644, which are herein incorporated by reference in their entireties.

[0371] 3. Certain GLP-1 peptide conjugate moieties

[0372] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide or a fragment or mutant thereof. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide or a fragment or mutant thereof. In certain embodiments, the oligonucleotide is a modified oligonucleotide. Any GLP-1 peptide or a fragment or mutant thereof known in the art can be used in several embodiments. In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide described in US20140206607; US 9,187,522; US 8,329,419; or WO 2007 / 124461, which is incorporated herein by reference in its entirety. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide described in US20140206607; US 9,187,522; US 8,329,419; or WO 2007 / 124461, which is incorporated herein by reference in its entirety.

[0373] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion comprising at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 30, or 31 consecutive amino acid portions: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, the conventional three-letter code of the amino acid sequence is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the same length portion of the amino acid sequence of GLP-1 (7-37): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (SEQ ID NO: 1), wherein NH2 indicates the C-terminal amide.

[0374] In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the same length portion of the amino acid sequence of GLP-1(7-37). , 29, 30, or 31 consecutive amino acid portions: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the same length portion of the amino acid sequence of GLP-1 (7-37): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (SEQ ID NO: 1), wherein NH2 indicates the C-terminal amide.

[0375] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of GLP-1(7-37). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of GLP-1 (7-37).

[0376] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1) throughout its entire length. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1) throughout its entire length.

[0377] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the entire length of the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the entire length of the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1).

[0378] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1).

[0379] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion consists of the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion consists of the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1).

[0380] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR-NH2, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2 (SEQ ID NO: 2). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2 (SEQ ID NO: 2). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1(7-36): HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2).In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the amino acid sequence of GLP-1(7-36): HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (SEQ ID NO: 2).

[0381] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the amino acid sequence of GLP-1 (7-36) amide (SEQ ID NO: 2). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the amino acid sequence of GLP-1 (7-36) amide (SEQ ID NO: 2). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the amino acid sequence of GLP-1 (7-36) (SEQ ID NO: 2). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the amino acid sequence of GLP-1 (7-36) (SEQ ID NO: 2).

[0382] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion comprising the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three letter code of the amino acid sequence is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion comprising the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), wherein NH2 indicates the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), wherein NH2 indicates the C-terminal amide.

[0383] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three-letter code of the amino acid sequence is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), wherein NH2 indicates the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, the conventional three-letter code of the amino acid sequence is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 3). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the following amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), wherein NH2 indicates the C-terminal amide.

[0384] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion comprising the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three letter code of the amino acid sequence is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion comprising the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), wherein NH2 indicates the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), wherein NH2 indicates the C-terminal amide.

[0385] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three-letter code of the amino acid sequence is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4). In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, the GLP-1 peptide conjugate portion consisting of the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), wherein NH2 indicates the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion consists of the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three-letter code of which is: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 4). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion consists of the following amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), wherein NH2 indicates the C-terminal amide.

[0386] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and an analog of a GLP-1 peptide conjugate moiety, including, but not limited to, liraglutide (from Novo Nordisk) ); Albiglutide (from GlaxoSmithKline ); tasiroxicam (Hoffman La-Roche); LY2189265 (Eli Lilly and Company)); LY2428757 (Eli Lilly); desamino-His7, Arg26, Lys34-((nε(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37); desamino-His7, Arg26, Lys34(nε-octanoyl)-GLP-1(7-37); Arg26,34, Lys38(Nε-(Ω-carboxypentadecanoyl))-GLP-1(7-38); Arg26,34, Lys36(Nε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-36); Aib8.35, Arg26,34, Phe31-GLP-1(7-36))(SEQ ID NO:5); HXaa8EGTFTSDVSSYLEXaa22Xaa23AAKEFIXaa30WLXaa33Xaa34G Xaa36Xaa37; wherein Xaa8 is A, V, or G; Xaa22 is G, K, or E; Xaa23 is Q or K; Xaa30 is A or E; Xaa33 is V or K; Xaa34 is K, N, or R; Xaa36 is R or G; and Xaa37 is G, H, P, or absent (SEQ ID NO:6); Arg34-GLP-1 (7-37) (SEQ ID NO:7); Glu30-GLP-1 (7-37) (SEQ ID NO:8); Lys22-GLP-1 (7-37) (SEQ ID NO:9); Gly8.36,Glu22-GLP-1(7-37)(SEQ ID NO:10); Val8,Glu22,Gly36-GLP-1(7-37)(SEQ ID NO:11); Gly8.36,Glu22,Lys33,Asn34-GLP-1(7-37)(SEQ ID NO:12); Val8,Glu22,Lys33,Asn34,Gly36-GLP-1(7-37)(SEQ ID NO:13); Gly8.36,Glu22,Pro37-GLP-1(7-37)(SEQ ID NO:14); Val8,Glu22,Gly36Pro37-GLP-1(7-37)(SEQ ID NO:15); Gly8.36,Glu22,Lys33,Asn34,Pro37-GLP-1(7-37)(SEQ ID NO:16); Val8,Glu22,Lys33,Asn34,Gly36Pro37-GLP-1(7-37)(SEQ ID NO:17); Gly8.36,Glu22-GLP-1(7-36) (SEQ ID NO: 18); Val8,Glu22,Gly36-GLP-1(7-36) (SEQ ID NO: 19); Val8,Glu22,Asn34,Gly36-GLP-1(7-36) (SEQ ID NO: 20); Gly8.36,Glu22,Asn34-GLP-1(7-36) (SEQ ID NO: 21). Any of the foregoing analogs may be optionally amidated. .

[0387] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and an analog of a GLP-1 peptide conjugate moiety, including but not limited to liraglutide, taserostat, exenatide, lixisenatide, semaglutide. These analogs are described in Lorenz M et al., "Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity," Bioorg Med Chem Lett. 2013 Jul 15; 23(14):4011-8, which is incorporated herein by reference in its entirety.

[0388] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: H-AibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO:22), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Cys (SEQ ID NO:22), wherein Aib is aminoisobutyric acid.

[0389] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: H-AibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSX-NH 2( SEQ ID NO: 23), wherein Aib is aminoisobutyric acid, X is penicillamine, and NH2 indicates the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

[0390] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC, the conventional three-letter code of which is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Cys (SEQ ID NO: 24). In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC-NH2 (SEQ ID NO:24), wherein NH2 indicates the C-terminal amide.

[0391] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 25). In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO:25), wherein H indicates the N-terminus and NH2 indicates the C-terminal amide.

[0392] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: AGEGTF TSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSC, the conventional three letter code of which is: Ala-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Ala-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Cys (SEQ ID NO: 26). In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: AGEGTF TSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO: 26), wherein NH2 indicates the C-terminal amide.

[0393] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: AGEGTF TSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSX, wherein X is penicillamine, the conventional three letter code of which is: Ala-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Ala-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Pen (SEQ ID NO: 27), wherein Pen is penicillamine. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: AGEGTFTSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSX-NH2 (SEQ ID NO: 27), wherein X is penicillamine and NH2 indicates the C-terminal amide.

[0394] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLV, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val (SEQ ID NO:28), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLV-NH2 (SEQ ID NO: 28), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0395] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVK, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (SEQ ID NO:29), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVK-NH2 (SEQ ID NO:29), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0396] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKG, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO:30), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO:30), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0397] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGG, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly (SEQ ID NO:31), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGG-NH2 (SEQ ID NO:31), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0398] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGP, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro (SEQ ID NO:32), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGP-NH2 (SEQ ID NO:32), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0399] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPS, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser (SEQ ID NO:33), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPS-NH2 (SEQ ID NO:33), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0400] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSS, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser (SEQ ID NO:34), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSS-NH2 (SEQ ID NO:34), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0401] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSG, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly (SEQ ID NO:35), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSG-NH2 (SEQ ID NO:35), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0402] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGA, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala (SEQ ID NO:36), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGA-NH2 (SEQ ID NO:36), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0403] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAP, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro (SEQ ID NO:37), wherein Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAP-NH2 (SEQ ID NO:37) wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0404] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSZ, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Zaa (SEQ ID NO: 38), wherein Z or Zaa is 4-azidonorleucine comprising:

[0405]

[0406] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSZ-NH2 (SEQ ID NO: 38), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0407]

[0408] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRGZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Zaa (SEQ ID NO: 39), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0409]

[0410] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRGZ-NH2 (SEQ ID NO: 39), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0411]

[0412] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEGQAANXEFIAWLVRGRG, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Asn-Xaa-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 40), wherein Aib is aminoisobutyric acid and X or Xaa is lysine (5-azidopentanoic acid amide) having the following formula:

[0413]

[0414] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEGQAANXEFIAWLVRGRG-NH2 (SEQ ID NO: 40), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and X or Xaa is lysine (5-azidopentanoic acid amide) having the following formula:

[0415]

[0416] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVK-AibRZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Aib-Arg-Zaa (SEQ ID NO: 41), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0417]

[0418] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVK-AibRZ-NH2 (SEQ ID NO:41), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0419]

[0420] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRZ, the conventional three letter code of which is: His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Zaa (SEQ ID NO: 42), wherein Z or Zaa is 4-azidonorleucine comprising:

[0421]

[0422] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRZ-NH2 (SEQ ID NO:42), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0423]

[0424] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Zaa (SEQ ID NO: 43), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0425]

[0426] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPZ-NH2 (SEQ ID NO: 43), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0427]

[0428] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Zaa (SEQ ID NO: 44), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0429]

[0430] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSZ-NH2 (SEQ ID NO: 44), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0431]

[0432] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Zaa (SEQ ID NO: 45), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0433]

[0434] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKZ-NH2 (SEQ ID NO: 45), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0435]

[0436] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Zaa (SEQ ID NO: 46), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0437]

[0438] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVZ-NH2 (SEQ ID NO: 46), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0439]

[0440] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVC, the conventional three-letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Cys (SEQ ID NO:47), wherein Aib is aminoisobutyric acid.

[0441] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVC-NH2 (SEQ ID NO:47), wherein Aib is aminoisobutyric acid and NH2 indicates the C-terminal amide.

[0442] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Zaa (SEQ ID NO: 48), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0443]

[0444] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLZ-NH2 (SEQ ID NO: 48), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0445]

[0446] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Zaa (SEQ ID NO: 49), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0447]

[0448] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWZ-NH2 (SEQ ID NO: 49), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0449]

[0450] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Zaa (SEQ ID NO: 50), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0451]

[0452] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAZ-NH2 (SEQ ID NO: 50), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0453]

[0454] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGZ, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Zaa (SEQ ID NO: 51), wherein Z or Zaa is 4-azidonorleucine comprising:

[0455]

[0456] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGZ-NH2 (SEQ ID NO:51), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0457]

[0458] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNZ, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Zaa (SEQ ID NO: 52), wherein Z or Zaa is 4-azidonorleucine comprising:

[0459]

[0460] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNZ-NH2 (SEQ ID NO: 52), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0461]

[0462] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKZ, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Zaa (SEQ ID NO: 53), wherein Z or Zaa is 4-azidonorleucine comprising:

[0463]

[0464] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKZ-NH2 (SEQ ID NO: 53), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0465]

[0466] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLZ, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Zaa (SEQ ID NO: 54), wherein Z or Zaa is 4-azidonorleucine comprising:

[0467]

[0468] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLZ-NH2 (SEQ ID NO: 54), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0469]

[0470] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWZ, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Zaa (SEQ ID NO: 55), wherein Z or Zaa is 4-azidonorleucine comprising:

[0471]

[0472] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWZ-NH2 (SEQ ID NO: 55), wherein NH2 indicates the C-terminal amide and Z or Zaa is 4-azidonorleucine comprising:

[0473]

[0474] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSZ, the conventional three letter code of which is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Zaa (SEQ ID NO: 56), wherein Aib is aminoisobutyric acid and Z or Zaa is 4-azidonorleucine comprising:

[0475]

[0476] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSZ-NH2 (SEQ ID NO: 56), wherein Aib is aminoisobutyric acid, NH2 indicates the C-terminal amide, and Z or Zaa is 4-azidonorleucine comprising:

[0477]

[0478] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises or consists of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC, the conventional three letter code of which is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 57).

[0479] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC-NH2 (SEQ ID NO:57), wherein NH2 indicates the C-terminal amide.

[0480] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0481] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the entire length of the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of any one of SEQ ID NOs: 1-57. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the entire length of the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0482] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the entire length of the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-57. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion of 8 to 50 amino acids in length, the entire length of the GLP-1 peptide conjugate portion being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0483] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof, when compared to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein the GLP-1 peptide conjugate portion comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof, when compared to the amino acid sequence of GLP-1 (7-37) (SEQ ID NO: 1).

[0484] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate portion comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1-57. In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate portion comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof when compared to the amino acid sequence of any one of SEQ ID NOs: 1-57. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof when compared to the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0485] In any of the above embodiments, the GLP-1 peptide conjugate portion may comprise conservative amino acid substitutions, amino acid analogs, or amino acid derivatives. In certain embodiments, conservative amino acid substitutions comprise replacing one aliphatic amino acid with another aliphatic amino acid; replacing serine with threonine, or replacing threonine with serine; replacing one acidic residue with another acidic residue; replacing one residue with an amide group with another residue with an amide group; exchanging one basic residue with another basic residue; or replacing one aromatic residue with another aromatic residue, or a combination thereof, and the aliphatic residue comprises alanine, valine, leucine, isoleucine, or a synthetic equivalent thereof; the acidic residue comprises aspartic acid, glutamic acid, or a synthetic equivalent thereof; the residue comprising an amide group comprises aspartic acid, glutamic acid, or a synthetic equivalent thereof; the basic residue comprises lysine, arginine, or a synthetic equivalent thereof; or the aromatic residue comprises phenylalanine, tyrosine, or a synthetic equivalent thereof.

[0486] Additional GLP-1 peptide conjugate moieties or analogs that can be used in the examples provided herein are described in US20140206607; US 9,187,522; WO 2007 / 124461; WO 2014 / 096179; WO 2009 / 030738; WO2016 / 055610; and US 8,329,419, which are incorporated herein by reference in their entirety.

[0487] Conjugate Linker

[0488] In certain embodiments, the conjugate linker connects the GLP-1 receptor ligand conjugate moiety to the oligonucleotide. In certain compounds, the GLP-1 receptor ligand conjugate moiety is attached to the oligonucleotide via a conjugate linker by a single bond. In certain embodiments, the conjugate linker comprises a chain structure (such as a hydrocarbon chain), or an oligomer of repeating units (such as ethylene glycol, nucleosides, or amino acid units).

[0489] In certain embodiments, the conjugate linker comprises one or more groups selected from the following: alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxyamino. In certain such embodiments, the conjugate linker comprises a group selected from the following: alkyl, amino, oxo, amide, and ether. 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 portion. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.

[0490] In certain embodiments, the conjugate linker (including the conjugate linker described above) is a bifunctional linking portion, such as those known in the art for attaching the conjugate group to the parent compound (such as oligonucleotides provided herein). Generally, the bifunctional linking portion comprises at least two functional groups. One of the functional groups is selected to be bonded to a specific site on the compound, and the other functional group is selected to be bonded to the conjugate group. Examples of functional groups used in the bifunctional linking portion include but are not limited to electrophilic reagents for reacting with nucleophilic groups and nucleophilic reagents for reacting with electrophilic groups. In certain embodiments, the bifunctional linking portion comprises one or more selected from the following groups: amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl and alkynyl.

[0491] Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic 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-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Alkynyl, wherein a non-limiting list of preferred substituent groups includes hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0492] In certain embodiments, the conjugate linker comprises 1-10 linkers-nucleosides. In certain embodiments, such linkers-nucleosides are modified nucleosides. In certain embodiments, such linkers-nucleosides comprise modified sugar moieties. In certain embodiments, linkers-nucleosides are unmodified. In certain embodiments, linkers-nucleosides comprise optionally protected heterocyclic bases selected from the following: purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable part is a nucleoside selected from the following: uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is generally desirable that linkers-nucleosides are cleaved from the compound after they reach the target tissue. Therefore, linkers-nucleosides are usually connected to each other and to the rest of the compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0493] Herein, it is not considered that the joint-nucleoside is a part of the oligonucleotide. Therefore, in the following examples, wherein the compound comprises an oligonucleotide consisting of a nucleoside connected by a specified number or range and / or a percentage complementarity specified with a reference nucleic acid, and the compound also comprises a conjugate group, the conjugate group comprises a conjugate joint, the conjugate joint comprises a joint-nucleoside, and those joints-nucleosides are not counted in the length of the oligonucleotide and are not used to determine the percentage complementarity of the oligonucleotide for the reference nucleic acid. For example, a compound may comprise a modified oligonucleotide consisting of (1) 8-30 nucleosides, and (2) a conjugate group comprising 1-10 joints-nucleosides, the joints-nucleosides being adjacent to the nucleosides of the modified oligonucleotide. The total number of nucleosides connected continuously in such a compound exceeds 30. Alternatively, a compound may comprise a modified oligonucleotide, the oligonucleotide consisting of 8-30 nucleosides and having no conjugate group. The total number of nucleosides connected continuously in such a compound does not exceed 30. Unless otherwise indicated, the conjugate joint comprises no more than 10 joints-nucleosides. In certain embodiments, the conjugate linker comprises no more than 5 linker-nucleosides. In certain embodiments, the conjugate linker comprises no more than 3 linker-nucleosides. In certain embodiments, the conjugate linker comprises no more than 2 linker-nucleosides. In certain embodiments, the conjugate linker comprises no more than 1 linker-nucleoside.

[0494] In certain embodiments, it is desirable that the conjugate group is cut from the oligonucleotide. For example, in some cases, the compound comprising a specific conjugate part is better absorbed by a specific cell type, but once the compound is absorbed, it is desirable that the conjugate group is cut to release unconjugated or parent oligonucleotide. Therefore, some conjugates may include one or more cleavable parts, typically in the conjugate joint. In certain embodiments, the cleavable part is a cleavable bond. In certain embodiments, the cleavable part is a group of atoms comprising at least one cleavable bond. In certain embodiments, the cleavable part includes a group of atoms having one, two, three, four or more than four cleavable bonds. In certain embodiments, the cleavable part is selectively cut in a cell or subcellular compartment (such as a lysosome). In certain embodiments, the cleavable part is selectively cut by an endogenous enzyme (such as a nuclease).

[0495] In certain embodiments, the cleavable bond is selected from: amide, ester, ether, one or two esters of phosphodiester, phosphate, carbamate or disulfide. In certain embodiments, the cleavable bond is one or two esters of phosphodiester. In certain embodiments, the cleavable portion comprises phosphate or phosphodiester. In certain embodiments, the cleavable portion is a phosphate ester connection between an oligonucleotide and a conjugate portion or a conjugate group.

[0496] In certain embodiments, the cleavable part comprises one or more joints-nucleosides or is composed of it.In certain such embodiments, one or more joints-nucleosides are connected to each other and / or connected to the rest of the compound by a cleavable bond.In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds.In certain embodiments, the cleavable part is a 2'-deoxynucleoside, which is attached to the 3' or 5'-terminal nucleoside of the oligonucleotide by a phosphate internucleoside connection and is covalently attached to the conjugate joint or the rest of the conjugate part by a phosphate or thiophosphate connection.In certain such embodiments, the cleavable part is a 2'-deoxyadenosine.

[0497] 1. Certain hexylamino linkers

[0498] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0499]

[0500] wherein each n is independently selected from 0, 1, 2, 3, 4, 5, 6, or 7.

[0501] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0502]

[0503] wherein each n is independently from 1 to 20; and p is from 1 to 6.

[0504] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0505]

[0506] wherein each n is independently from 1 to 20.

[0507] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0508]

[0509] wherein each n is independently from 1 to 20.

[0510] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0511]

[0512] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0513]

[0514] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker selected from the following structures:

[0515]

[0516] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker having the structure:

[0517]

[0518] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker having the structure:

[0519] in

[0520] X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and

[0521] Y is directly or indirectly attached to the modified oligonucleotide.

[0522] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via any of the conjugate linkers described in WO 2014 / 179620, which is incorporated herein by reference in its entirety.

[0523] 2. Certain alkyl phosphate linkers

[0524] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker having the structure:

[0525]

[0526] in:

[0527] The phosphate group is linked to the modified oligonucleotide, and Y is linked to the conjugate group;

[0528] Y is a phosphodiester or amino (-NH-) group;

[0529] Z is a pyrrolidinyl group having the formula:

[0530]

[0531] j is 0 or 1;

[0532] n is from about 1 to about 10;

[0533] p is from 1 to about 10;

[0534] m is 0 or from 1 to 4; and

[0535] When Y is amino, m is 1.

[0536] In certain embodiments, Y is an amino group (-NH-). In certain embodiments, Y is a phosphodiester group. In certain embodiments, n is 3 and p is 3. In certain embodiments, n is 6 and p is 6. In certain embodiments, n is from 2 to 10 and p is from 2 to 10. In certain embodiments, n and p are different. In certain embodiments, n and p are the same. In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, j is 0. In certain embodiments, j is 1 and Z has the formula:

[0537]

[0538] In certain embodiments, wherein n is 2 and p is 3. In certain embodiments, n is 5 and p is 6.

[0539] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker having the structure:

[0540]

[0541] wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and

[0542] wherein T1 comprises the modified oligonucleotide; and Bx is a modified or unmodified nucleobase.

[0543] 3. Certain click chemistry linkers

[0544] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker is prepared using click chemistry known in the art. Click chemistry has been used to prepare compounds in which alkynylphosphonate internucleoside linkages on an oligomeric compound attached to a solid support are converted to 1,2,3-triazolephosphonate internucleoside linkages and then cleaved from the solid support (Krishna et al., J. Am. Chem. Soc. 2012, 134(28), 11618-11631), which is incorporated herein by reference in its entirety. Additional linkers suitable for several embodiments can be prepared by click chemistry as described in "Click Chemistry for Biotechnology and Materials Science," Joerg Laham, ed., Wiley 2009, which is incorporated herein by reference in its entirety.

[0545] In certain embodiments, a click reaction can be used to link the GLP-1 receptor ligand conjugate moiety and the oligonucleotide by reacting with:

[0546]

[0547] Oligonucleotides with terminal amines include, but are not limited to, the following compounds:

[0548]

[0549] wherein Y is directly or indirectly attached to the oligonucleotide to yield:

[0550]

[0551] It can react with the GLP-1 receptor ligand conjugate moiety having an azide to yield:

[0552]

[0553] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety.

[0554] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker is prepared from:

[0555]

[0556] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises:

[0557]

[0558] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises:

[0559]

[0560] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0561]

[0562] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; and

[0563] Y is attached directly or indirectly to the oligonucleotide.

[0564] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0565]

[0566] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; and

[0567] Y is attached directly or indirectly to the oligonucleotide.

[0568] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0569]

[0570] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; and

[0571] Y is attached directly or indirectly to the oligonucleotide.

[0572] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker is prepared using click chemistry and a disulfide bond.

[0573] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0574]

[0575] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety;

[0576] n and o are independently selected from 2 to 10; and

[0577] Y is attached directly or indirectly to the oligonucleotide.

[0578] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0579]

[0580] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety;

[0581] n, o and p are independently selected from 2 to 10;

[0582] m is 0 or 1; and

[0583] Y is attached directly or indirectly to the oligonucleotide.

[0584] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0585]

[0586] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety;

[0587] m is 0 or 1; and

[0588] Y is attached directly or indirectly to the oligonucleotide.

[0589] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0590]

[0591] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety;

[0592] m is 1; and

[0593] Y is attached directly or indirectly to the oligonucleotide.

[0594] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises:

[0595]

[0596] wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety;

[0597] n and o are independently selected from 2 to 10; and

[0598] Y is attached directly or indirectly to the oligonucleotide.

[0599] 4. Certain maleimide and maleimide acid linkers

[0600] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises:

[0601]

[0602] X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and

[0603] Y is attached directly or indirectly to the oligonucleotide.

[0604] In certain embodiments, the above conjugate linker can connect the peptide to the oligonucleotide. In certain embodiments, the compound comprises an oligonucleotide connected to the peptide via a conjugate linker, wherein the conjugate linker comprises:

[0605]

[0606] X is directly or indirectly attached to the peptide; and

[0607] Y is attached directly or indirectly to the oligonucleotide.

[0608] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0609]

[0610] Where R=(CH2) n And n is from 1 to 12;

[0611] X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0612] Y is attached directly or indirectly to the oligonucleotide.

[0613] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0614]

[0615] in

[0616] m is from 1 to 12;

[0617] X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0618] Y is attached directly or indirectly to the oligonucleotide.

[0619] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, wherein the first compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0620]

[0621] Where R=(CH2) n And n is from 1 to 12;

[0622] X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0623] Y is directly or indirectly attached to said oligonucleotide;

[0624] and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0625]

[0626] Where R=(CH2) n And n is from 1 to 12;

[0627] X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0628] Y is attached directly or indirectly to the oligonucleotide.

[0629] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, wherein the first compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0630]

[0631] in

[0632] m is from 1 to 12;

[0633] X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0634] Y is directly or indirectly attached to said oligonucleotide;

[0635] and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0636]

[0637] in and m is from 1 to 12;

[0638] X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0639] Y is attached directly or indirectly to the oligonucleotide.

[0640] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0641]

[0642] wherein X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0643] Y is attached directly or indirectly to the oligonucleotide.

[0644] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0645]

[0646] wherein X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0647] Y is attached directly or indirectly to the oligonucleotide.

[0648] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, wherein the first compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0649]

[0650] wherein X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0651] Y is directly or indirectly attached to said oligonucleotide;

[0652] and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety (e.g., a peptide) via a conjugate linker, wherein the conjugate linker comprises:

[0653]

[0654] wherein X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety, e.g., a peptide; and

[0655] Y is attached directly or indirectly to the oligonucleotide.

[0656] 5. Certain disulfide bonds

[0657] In certain embodiments, the compound comprises an oligonucleotide connected to a GLP-1 receptor ligand conjugate portion via a conjugate linker, wherein the conjugate linker comprises a disulfide bond. In certain embodiments, the oligonucleotide comprises an active disulfide that forms a disulfide bond with the GLP-1 peptide conjugate portion. In certain embodiments, the compound comprises an oligonucleotide comprising an active disulfide portion that can form a cleavable or reversible bond with the GLP-1 peptide conjugate portion. In certain embodiments, the compound comprises an oligonucleotide that is directly attached to the GLP-1 peptide conjugate portion via a disulfide bond (without a conjugate linker).

[0658] In certain embodiments, the compound comprises a linker between an oligonucleotide and an active disulfide moiety. In another embodiment, the active disulfide moiety has the formula -SS(O)2-substituted or unsubstituted C1-C12 alkyl or -SSC(O)O-substituted or unsubstituted C1-C12 alkyl. Preferred active disulfide moieties are thiomethanesulfonate and dithiomethylester groups. In further embodiments, the active disulfide is a substituted or unsubstituted dithiopyridyl, a substituted or unsubstituted dithiobenzothiazolyl, or a substituted or unsubstituted dithiotetrazolyl. Preferred reactive disulfides are 2-dithiopyridyl, 2-dithio-3-nitropyridyl, 2-dithio-5-nitropyridyl, 2-dithiobenzothiazolyl, N-(C1-C12 alkyl)-2-dithiopyridyl, 2-dithiopyridyl-N-oxide, or 2-thio-1-methyl-1H-tetrazolyl.

[0659] In some embodiments, the active disulfide moiety has the formula -SS(O) n -R1, where

[0660] n is 0, 1, or 2; and

[0661] R1 is selected from substituted or unsubstituted heterocycle, substituted or unsubstituted aliphatic, or -C(O)O-R2, wherein R2 is substituted or unsubstituted aliphatic.

[0662] In another embodiment, the active disulfide moiety has the formula -SS(O)2 substituted or unsubstituted C1-C 12 Alkyl or -SSC(O)O-substituted or unsubstituted C1-C 12In certain embodiments, the active disulfide moiety includes thiomethanesulfonate and dithiomethyl ester. In other embodiments, the active disulfide can be a substituted or unsubstituted dithiopyridyl, a substituted or unsubstituted dithiobenzothiazolyl, or a substituted or unsubstituted dithiotetrazolyl. Other examples of active disulfides include, but are not limited to, 2-dithiopyridyl, 2-dithio-3-nitropyridyl, 2-dithio-5-nitropyridyl, 2-dithiobenzothiazolyl, N-(C1-C 12 alkyl)-2-dithiopyridyl, 2-dithiopyridyl-N-oxide, and 2-dithio-1-methyl-1H-tetrazolyl.

[0663] In some embodiments, the divalent linking group is a divalent substituted or unsubstituted aliphatic group. In another embodiment, the divalent linking group has the formula -Q1-G-Q2-, wherein

[0664] Q1 and Q2 are independently absent or selected from substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted alkylene or -(CH2) m -O-(CH2) p -,in

[0665] Each m and p is independently an integer from 1 to about 10;

[0666] G is -NH-C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -NH-C(S)-NH-, -NH-O-, NH-C(O)-O-, or -O-CH2-C(O)-NH-.

[0667] Examples of divalent linking groups include, but are not limited to:

[0668]

[0669] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 peptide conjugate moiety via a disulfide bond as described in US 7,713,944, which is incorporated herein by reference in its entirety. In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 peptide conjugate moiety, wherein the oligonucleotide comprises an activated disulfide as described in US 7,713,944, which is incorporated herein by reference in its entirety.

[0670] In certain embodiments, any of the above compounds comprising an oligonucleotide linked to a GLP-1 peptide conjugate portion by a disulfide bond (directly or through a conjugate linker described herein) may comprise a disulfide bond between a cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid portion of the GLP-1 peptide conjugate portion and the oligonucleotide or conjugate linker. In certain embodiments, the compound comprises an oligonucleotide directly linked to a GLP-1 peptide conjugate portion by a disulfide bond. In certain embodiments, the compound comprises an oligonucleotide directly linked to a GLP-1 peptide conjugate portion by a disulfide bond, wherein the disulfide bond is between the oligonucleotide and the cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid portion of the GLP-1 peptide conjugate portion. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein a disulfide bond connects the conjugate linker and the GLP-1 peptide conjugate portion, and the oligonucleotide is attached to the conjugate linker. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate portion, wherein a disulfide bond connects the conjugate linker to a cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid portion of the GLP-1 peptide conjugate portion, and the oligonucleotide is attached to the conjugate linker. In certain embodiments, the cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid portion is located at the N-terminus, C-terminus, side chain, or internal amino acid position of the GLP-1 peptide conjugate portion.

[0671] 6. Ligands that can be cleaved by certain enzymes

[0672] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises an enzyme-cleavable moiety. In certain embodiments, the GLP-1 receptor ligand conjugate moiety is a GLP-1 peptide conjugate moiety. In certain embodiments, the enzyme-cleavable moiety is a peptide, such as a dipeptide.

[0673] Enzymes known in the art for activating prodrugs can be used to cleave enzyme-cleavable moieties provided in certain embodiments. In certain embodiments, the enzyme-cleavable moiety can be cleaved by DT diaphorase, plasmin, carboxypeptidase G2, thymidine kinase (viral), cytosine deaminase, glucose oxidase, xanthine oxidase, carboxypeptidase A, α-galactosidase, β-glucosidase, azoreductase, γ-glutamyl transferase, β-glucuronidase, β-lactamase, alkaline phosphatase, aminopeptidase, penicillin amidase, or nitroreductase.

[0674] In certain embodiments, the enzyme-cleavable portion is cleavable by a protease or peptidase. In certain embodiments, the enzyme-cleavable portion is cleavable by a protease or peptidase selected from the group consisting of pepsin, pepsin-2, chymosin, renin, renin-2, cathepsin D, cathepsin E, penicillin pepsin, rhizopus pepsin, mucorpepsin, barrier pepsin, aspergillus pepsin I, endothiapepsin, saccharopepsin, phytepsin, plasmodium pepsin-1, plasmodium pepsin-2, yeast asparagine-1, yeast asparagine-2, pitcher plant protease, pepsin-1, napsin A. HIV-1 retropepsin, HIV-2 retropepsin, simian immunodeficiency virus retropepsin, equine infectious anemia virus retropepsin, feline immunodeficiency virus retropepsin, murine leukemia virus type retropepsin, Mason-Pfizer leukemia virus type retropepsin, human endogenous retrovirus K retropepsin, retropepsin (human T-cell leukemia virus), bovine leukemia virus retropepsin, Rous sarcoma virus retropepsin, scytalidoglutamic peptidase, aspergilloglutamic peptidase, thermolysin, signal peptidase II, spumapepsin, type 4 prepilinpeptidase1), omptin, plasminogen activator Pla, papain, chymopapain, papaya cysteine ​​protease (caricain), aminoacetyl endopeptidase, pineapple enzyme stem bromelain, fig proteinase, kiwi proteinase, cathepsin V, vignain, cathepsin X, zingipain, cathepsin F, ananain, pineapple fruit proteinase, cathepsin L, cathepsin L1 (flavour species), cathepsin S, cathepsin K, Cathepsin H, aleurain, histolysain, cathepsin B, dipeptidyl peptidase I, peptidase 1 (mite), CPB peptidase, cruzipain, cathepsin peptidase (V-cathpeptidase), bleomycin hydrolase (animal), bleomycin hydrolase (yeast), aminopeptidase C, CPC peptidase, calpain-1, calpain-2, calpain-3, Tpr peptidase (Porphyromonas gingivalis), poliovirus-type histolysin 3C ( picornain 3C), hepatitis A virus-type picornain 3C, poliovirus-type picornain 3C, human rhinovirus 2-type picornain 3C, foot-and-mouth disease virus picornain 3C, enterovirus picornain 2A, rhinovirus picornain 2A, nuclear inclusion-a peptidase (plum pox virus), tobacco etch virus NIa peptidase, adenain, potato virus Y-type peptidase accessory component, sindbis virus-type nsP2 peptidase, streptopain, clostripain, ubiquitin hydrolase-L1hydrolase-L1), ubiquitin hydrolase-L3, legumin (plant β form), legumin, animal-type, caspase-1, caspase-3, caspase-7, caspase-6, caspase-8, caspase-9, pyroglutamyl-peptidase I (prokaryotes), pyroglutamyl-peptidase I (chordates), papain-like peptidase 1 of murine hepatitis corona virus, ubiquitin-specific protease 5, turnip yellow mosaic virus group protease, rabbit hemorrhagic disease virus 3C-like protease, gingipain RgpA, gingipain Kgp, gamma-glutamine hydrolase, foot-and-mouth disease virus L-protease, porcine transmissible gastroenteritis virus-type major peptidase, calicivirin, staphopain A A), Ulp1 peptidase, separase (yeast-type), YopJ protein, PfpI peptidase, sortase A (staphylococcus-type), aminopeptidase N, lysyl aminopeptidase (bacterial), aminopeptidase A, leukotriene A4 hydrolase, pyroglutamyl-peptidase II, cytosolic alanyl aminopeptidase, cystine peptidase, aminopeptidase B, aminopeptidase Ey, angiotensin converting enzyme peptidase unit 1, peptidyl dipeptidase Acer, angiotensin converting enzyme peptidase unit 2, angiotensin converting enzyme-2, thimet oligosaccharide oligopeptidase oligopeptidase), neurolysin, saccharolysin, oligopeptidase A, peptidyl dipeptidase Dcp, mitochondrial intermediate peptidase, oligopeptidase F, thermolysin, vibriolysin, pseudolysin, coccolysin, aureolysin, stearolysin, mycolysin, snapalysin, leishmanin, bacterial collagenase V, bacterial collagenase G / A, matrix metalloproteinase-1, matrix metalloproteinase-8, matrix metalloproteinase-2, matrix metalloproteinase -9, matrix metalloproteinase-3, matrix metalloproteinase-10 (Homo sapiens-type), matrix metalloproteinase-11, matrix metalloproteinase-7, matrix metalloproteinase-12, envelopein, matrix metalloproteinase-13, membrane-type matrix metalloproteinase-1, membrane-type matrix metalloproteinase-2, matrix metalloproteinase-20, fragile protein (fragilysin), matrix metalloproteinase-26, serralysin, aeruginolysin, gametolysin, astaxanthin, meprin alpha subunit, procollagen C-peptidase, choriolysin LL), choriolysin H, flavastacin, fibrinolytic enzyme, jararhagin, disintegrin, rattlesnake metalloendopeptidase A, rattlesnake metalloendopeptidase B, rattlesnake metalloendopeptidase C, rattlesnake metalloendopeptidase E, atroxase, russellysin, ADAM1 peptidase, ADAM9 peptidase, ADAM10 peptidase, Kuzbanian peptidase (non-mammalian), ADAM12 peptidase, ADAM17 peptidase, ADAMTS4 peptidase, ADAMTS1 peptidase, ADAMTS5 peptidase, ADAMTS13 peptidase, procollagen I N-peptidase, enkephalinase, endothelin-converting enzyme 1, oligopeptidase O1, enkephalinase-2, PHEX peptidase, carboxypeptidase A1, carboxypeptidase A2, carboxypeptidase B, carboxypeptidase N, carboxypeptidase E, carboxypeptidase M, carboxypeptidase T, carboxypeptidase B2, carboxypeptidase A3, metallocarboxypeptidase D peptidase unit 1, metallocarboxypeptidase D peptidase unit 2, zinc D-Ala-D-Ala carboxypeptidase (Streptomyces-type), vanY D-Ala-D-Ala carboxypeptidase, vanX D-Ala-D-Ala dipeptidase, vasopressin, trypsin, mitochondrial processing peptidase β-subunit, phenelzine lyase, leucine aminopeptidase 3, leucyl aminopeptidase (plant-type), aminopeptidase I, aspartyl aminopeptidase, membrane dipeptidase, glutamate carboxypeptidase, peptidase T, carboxypeptidase Ss1, β-lysozygous metallopeptidase, staphylolysin, lysostaphin, methionyl aminopeptidase 1 (Escherichia-type), methionyl aminopeptidase 2, Xaa-Pro dipeptidase (bacterial-type), aminopeptidase P (bacterial), aminopeptidase P2, Xaa-Pro dipeptidase (eukaryotic), IgA1-specific metallopeptidase, aspartate hemolysin (tentoxilysi n), botulinum toxin (bontoxilysin), aminopeptidase Y, aminopeptidase Ap1, aminopeptidase S (Streptomyces-type), glutamate carboxypeptidase II, carboxypeptidase Taq, anthrax lethal factor, botulinum toxin, peptidyl-Lys metallopeptidase, FtsH peptidase, m-AAA peptidase, i-AAA peptidase, AtFtsH2 peptidase, pappa hair-1, Ste24 peptidase, dipeptidyl-peptidase III, site 2 peptidase, sporulation factor SpoIVFB, HybD peptidase, gpr peptidase, chymotrypsin A (bovine-type), granzyme B (Homo sapiens-type), factor VII-activated peptidase, trypsin (Streptomyces griseus-type), hypodermin C (hypoderminC), elastase-2, cathepsin G, myeloid cytokines, granzyme A, granzyme M, chymase (Homo sapiens-type), mast cell peptidase 1 (rat-type), duodenase, tryptase α, granzyme K, mast cell peptidase 5 (rat number), trypsin 1, chymotrypsin B, elastase-1, pancreatic endopeptidase E, pancreatic elastase II, enteropeptidase, chymotrypsin C, prostatic protein, kallikrein 1, kallikrein-related protease 2, kallikrein-related protease 3 , kallikrein 1 (mouse), kallikrein 1-related protease b3, kallikrein 1-related protease c2 (rat), kallikrein 13 (mouse), ancrod, prothrombin (bothrombin), complement factor D, complement component activated C1r, complement component activated C1s, complement factor Bb, mannan-binding lectin-related serine peptidase 1, complement factor I, coagulation factor XIIa, plasma kallikrein, coagulation factor XIa, coagulation factor IXa, coagulation factor VIIa, coagulation factor X a, thrombin, protein C (activated), thrombin C (Hippocampus, Limulus), activated coagulation factor B (Hippocampus, Limulus), activated chymosin (Limulus-type), spermase, hepsin, mannan-binding lectin-related serine peptidase 2, urokinase-type plasminogen activator, t-plasminogen activator, plasmin, kallikrein-related peptidase 6, plasminogen activator (Vampire bat-type), kallikrein-related peptidase 8, kallikrein-related peptidase 4, streptolysin A, streptolysin B, streptolysin E, α-cleaving endopeptidase, glutamyl peptidase I, DegP peptidase, HtrA2 peptidase, lysyl endopeptidase (bacterial), kallikrein-related peptidase 7, matriptase, togavirin, IgA1-specific serine peptidase (Neisseria-type), flavivirin, subtilisin Carlsberg, subtilisin lentus, thermophilic protease, subtilisin Ak1, lactocepin I, C5a peptidase, dentilisin, subtilisin BPN', subtilisin E, aqualysin 11), cerevisin, oryzin, endopeptidase K, thermomycolin, site-1 peptidase, kexin, furin, PCSK1 peptidase, PCSK2 peptidase, PCSK4 peptidase, PCSK6 peptidase, PCSK5 peptidase, PCSK7 peptidase, tripeptidyl-peptidase II, cucumisin, prolyl oligopeptidase, dipeptidyl-peptidase IV (eukaryotic organisms), acyl-peptidase, fibroblast activation protein α subunit, oligopeptidase B, carboxypeptidase Y, serine carboxypeptidase A, serine carboxypeptidase C, serine carboxypeptidase D, stem carboxypeptidase (kex carboxypeptidase), D-Ala-D-Ala carboxypeptidase A, K15-type DD-transpeptidase, D-Ala-D-Ala carboxypeptidase B, aminopeptidase DmpB, D-Ala-D-Ala peptidase C, peptidase Clp (type 1), Xaa-Pro dipeptidyl-peptidase, Lon-A peptidase, PIM1 peptidase, cytomegalovirus cytomegalovirus, herpesvirus 8-type cytomegalovirus, repressor LexA, UmuD protein, signal peptidase I, mitochondrial inner membrane peptidase 1, signal peptidase SipS, signal enzyme (animal) 21kDa component, lysosomal Pro-Xaa carboxypeptidase, dipeptidyl-peptidase II, hepacivirin, potyvirus P1 peptidase peptidase), pestivirus NS3 polyprotein peptidase, equine arteritis virus serine peptidase, prolyl aminopeptidase, C-terminal processing peptidase-1, C-terminal processing peptidase-2, tricorn core peptidase (Archaea), signal peptide peptidase A, infectious pancreatic necrosis birnavirus Vp4 peptidase (infectiouspancreatic necrosis birnavirus Vp4 peptidase), dipeptidase E, sedolisin, sedolisin-B, tripeptidyl-peptidase I, kumamolisin, physarolisin, SpoIVB peptidase, archaean proteasome, beta module, bacterial proteasome, beta module, HslV module of HslUV peptidase, constitutive proteasome catalytic subunit 1, constitutive proteasome catalytic subunit 2, constitutive proteasome catalytic subunit 3, gamma-glutamyltransferase 1 (bacterial-type), murein tetrapeptidase LD-carboxypeptidase (Escherichia-type), PepA aminopeptidase, presenilin 1, polyporopepsin, canditropsin, candidapepsin SAP2In some embodiments, the enzyme-cleavable portion can be cleaved by a cathepsin or a peptidase.

[0675] Compositions and methods for preparing pharmaceutical compositions

[0676] The compounds described herein may be mixed with pharmaceutically acceptable active or inert substances to prepare pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on a number of criteria, including but not limited to the route of administration, the extent of the disease, or the dose to be administered.

[0677] Certain embodiments provide pharmaceutical compositions comprising one or more compounds or salts thereof. In certain embodiments, pharmaceutical compositions comprise compounds described herein and a pharmaceutically acceptable diluent or carrier. In certain embodiments, pharmaceutical compositions comprise sterile saline solutions and one or more compounds described herein. In certain embodiments, such pharmaceutical compositions consist of sterile saline solutions and one or more compounds. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, pharmaceutical compositions comprise one or more compounds described herein and sterile water. In certain embodiments, pharmaceutical compositions consist of a compound described herein and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, pharmaceutical compositions comprise one or more compounds described herein and phosphate buffered saline (PBS). In certain embodiments, pharmaceutical compositions consist of one or more compounds described herein and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS.

[0678] Pharmaceutical compositions comprising the compounds described herein encompass any pharmaceutically acceptable salt, ester, or salt of such ester, or any other oligonucleotide that is capable of providing (directly or indirectly) a biologically active metabolite or residue thereof after being administered to an animal (including a human). Certain embodiments relate to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of the compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0679] NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

[0680] Although certain compounds, compositions, and methods described herein have been specifically described in terms of certain embodiments, the following examples are merely illustrative of the compounds described herein and are not intended to limit them.

[0681] Each reference cited herein, including but not limited to scientific articles, patent publications, GenBank Accession Numbers, etc., is incorporated herein by reference in its entirety.

[0682] Although the sequence table attached to this document identifies each sequence as "RNA" or "DNA" as needed, in fact those sequences can be modified with any combination of chemical modifications. Those skilled in the art will readily appreciate that it is arbitrary in some cases to describe modified oligonucleotides such as "RNA" or "DNA" under such names. For example, an oligonucleotide comprising a nucleoside (the nucleoside comprises a 2'-OH sugar moiety and a thymine base) can be described as a DNA with a modified sugar (2'-OH replaces a 2'-H of DNA) or an RNA with a modified base (thymine (methylated uracil) replaces the uracil of RNA). Therefore, the nucleotide sequences provided herein (including but not limited to those in the sequence table) 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. As a further example and not limitation, an oligomeric compound having a nucleobase sequence "ATCGATCG" encompasses any oligomeric compound having such a nucleobase sequence, whether modified or unmodified, including but not limited to such compounds comprising RNA bases, such as those having the sequence "AUCGAUCG" and those having some DNA bases and some RNA bases such as "AUCGATCG," as well as oligomeric compounds having other modified nucleobases, such as "ATmCGAUCG," where mC indicates a cytosine base comprising a methyl group at the 5-position.

[0683] Compounds described herein include (R) or (S), such as α or β, such as sugar anomers, or (D) or (L), such as amino acids, etc. All such possible isomers are included in the compounds provided herein, unless otherwise specified, including racemic and optically pure forms thereof. Likewise, all cis- and trans-isomers and tautomeric forms are also included. Compounds described herein include chiral pure or enriched mixtures and racemic mixtures. For example, oligonucleotides having multiple thiophosphate nucleoside connections include such compounds, wherein the chirality of the thiophosphate nucleoside connections is controlled or random.

[0684] Unless otherwise indicated, any compound described herein (including oligomeric compounds) includes pharmaceutically acceptable salts thereof.

[0685] The compounds described herein include variants in which one or more atoms are replaced with non-radioactive isotopes or radioactive isotopes of the specified elements. For example, a compound herein containing a hydrogen atom encompasses a 1 All possible deuterium substitutions of H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but are not limited to: 2 H or 3 H instead 1 H. 13 C or 14 C instead 12 C. 15 N instead 14 N. 17 O or 18 O instead 16 O. and 33 S. 34 S. 35 S or 36 S instead 32 S.

[0686] Examples

[0687] Example 1: Preparation of antisense oligonucleotides (ASOs) targeting MALAT1 conjugated to GLP-1 peptide

[0688] Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated via a 3-mercaptopropionate linker.

[0689] Unless otherwise stated, all reagents and solutions used to synthesize oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid supports are used to incorporate nucleoside residues, including, for example, T, A, G, and mC residues. For 2'-deoxyribonucleosides, cEt BNA nucleosides, and appropriately protected 6-amino-hexanol, 0.1M phosphoramidite in anhydrous acetonitrile solution is used.

[0690]

[0691] A 5'-hexylamino modified oligonucleotide (ISIS786434) (nucleobase sequence: TCAGCATTCTAATAGCAGC (SEQ ID NO: 38) was synthesized and purified using a standard solid phase oligonucleotide procedure. The 5' end of the modified oligonucleotide contained a hexamethylene linker and a terminal amine. Compound 1 (3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) was obtained from Chem-Impex (catalog #11566). The modified oligonucleotide (approximately 6 μmol) was dissolved in 125 μL sodium phosphate buffer at pH 8, and 12 μmol of compound 1 was dissolved in DMF. The solution of compound 1 was added dropwise to the modified oligonucleotide solution and allowed to react at room temperature. After 2-3 hours, the reaction was complete and the product 2 was purified by HPLC on a source 30Q resin using buffer A 100 mM NH4OAc / 30% ACN / H2O and buffer B 100 mM NH4OAc / 30% ACN / H2O+1.5M NaBr was purified by HPLC and desalted by HPLC on a reverse phase column. Product fractions were concentrated and stored at -20 °C.

[0692] Compound 2 was used as the starting material for the reaction with the GLP-1 peptide HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleA laTrpLeuValLysGlyGlyProSerSerAlaProProProSerCys-NH2 (SEQ ID NO: 22), which was synthesized via standard solid phase peptide synthesis. Aib is 2-aminoisobutyric acid. Compound 2 was dissolved in degassed water and 0.1M NaHCO3 was added to adjust the pH to about 8.0. The GLP-1 peptide was dissolved in 50 / 500.1M NaHCO3 (pH 8):DMF (dimethylformamide). The peptide solution was added to compound 2 in small portions (30% of the total volume each time) at 5 min intervals. After about 1 hr, the reaction mixture was diluted with water (5 times the volume of the reaction solution V / V) and the product was purified by HPLC on source 30Q resin with buffer A 100 mM NH4OAc / 30% ACN / H2O and buffer B 100 mM NH4OAc / 30% ACN / H2O+1.5 M NaBr. The product fractions were desalted by HPLC on a reverse phase column to yield ISIS 816385.

[0693] Example 2: Preparation of antisense oligonucleotides (ASOs) targeting MALAT1 conjugated to GLP-1 peptide

[0694] Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated to a C-terminal penicillamine via a 3-mercaptopropionate linker.

[0695]

[0696] Compound 2 was synthesized as described in Example 1 and used as a starting material for reaction with the GLP-1 peptide: HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleA laTrpLeuValLysGlyGlyProSerSerAlaProProProSerPen-NH2 (SEQ ID NO: 23), which was synthesized via standard solid phase peptide synthesis. Aib is 2-aminoisobutyric acid and Pen is penicillamine. Compound 2 was dissolved in degassed water and 0.1M NaHCO3 was added to adjust the pH to about 8.0. The GLP-1 peptide was dissolved in degassed water. The solution of compound 2 and the peptide solution were mixed under gentle vortexing and the pH was checked. 0.1M NaHCO3 was added to adjust the pH to about 7.5. After about 2 hr, additional peptide was added and NaHCO3 was added to adjust the pH to rise. The reaction was transferred to 4°C for approximately 65 hours and the product was purified by HPLC as described in Example 1.

[0697] Example 3: Preparation of antisense oligonucleotides (ASOs) targeting FOXO1 conjugated to GLP-1 peptide

[0698] Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated via a 3-mercaptopropionate linker.

[0699] The 5'-GLP-1 peptide-conjugated ASO (ION 913193) targeting FOXO1 was prepared according to the procedure of Example 1, and the oligonucleotide (ION 913192) modified with 5'-hexylamino group (nucleobase sequence:

[0700] TCATCTTCTTAAAATACCC (SEQ ID NO:59), starting with the chemical modifications: Tdo mCdo Ado Tks mCksTds Tds mCds Tds Tds Ads Ads Ads Aks Tes Aks mCes mCks mCk (k = cEt; d = 2'-deoxy; e = 2'-MOE; mC = 5-methylcytosine; o = phosphodiester; and s = phosphorothioate).

[0701] ION 913195, a control 5'-GLP-1 peptide-conjugated ASO with a nucleobase sequence mismatched with FOXO1, was prepared according to the procedure of Example 1 starting with a 5'-hexylamino-modified oligonucleotide (ION 913194) (nucleobase sequence: TCAGGCCAATACGCCGTCA (SEQ ID NO:60) with chemical modifications: Tdo mCdo Ado Gks Gks mCks mCdsAds Ads Tds Ads mCds Gds mCds mCds Gds Tks mCks Ak (k=cEt; d=2'-deoxy; e=2'-MOE; mC=5-methylcytosine; o=phosphodiester; and s=phosphorothioate).

[0702] Example 4: Preparation of antisense oligonucleotides (ASOs) targeting insulin conjugated to GLP-1 peptide

[0703] Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated via a mercaptopropionate linker.

[0704] The insulin-targeted 5′-GLP-1 peptide-conjugated ASO (ION 919553) was prepared according to the procedure of Example 1, using a 5′-hexylamino-modified oligonucleotide (ION 919553) (nucleobase sequence:

[0705] TCAGCCAAGGTCTGAAGGTCACC (SEQ ID NO: 61), with chemical modification:

[0706] Start with Tdo mCdo Ado Ges mCes mCes Aes Aes Gds Gds Tds mCds Tds Gds Ads AdsGds Gds Tes mCes Aes mCes mCe (k=cEt; d=2'-deoxy; e=2'-MOE; mC=5-methylcytosine; o=phosphodiester; and s=phosphorothioate).

[0707] Example 5: Preparation of antisense oligonucleotide (ASO) duplex targeting MALAT1 conjugated to GLP-1 peptide

[0708] ION 951976 (nucleobase sequence: GCTGCTATTAGAATGC (SEQ ID NO: 62) with chemical modifications: Ges mCeo Tdo Gdo mCdo Tdo Ado TdoTdo Ado Gdo Ado Ado Tds Ges mCe (d = 2'-deoxy; e = 2'-MOE; mC = 5-methylcytosine; o = phosphodiester; and s = phosphorothioate) was synthesized and purified using standard solid phase oligonucleotide procedures. ISIS 816385 described in Example 1 was hybridized to ION 951976 to generate a duplex of the two oligonucleotides.

[0709] Example 6: GLP-1 peptide-conjugated ASO specifically targets pancreatic β islet cells in vivo Study 1

[0710] To determine whether conjugation of GLP-1 peptides to ASOs increases ASO delivery to the pancreas, male C57BL / 6 mice fed a chow diet received 2 intravenous injections of 3-10-3cEt ASO (ISIS 556089) targeting murine MALAT1 (nucleobase sequence: GCATTCTAATAGCAGC) (SEQ ID NO: 63) or GLP-1 conjugated MALAT1 ASO (ISIS 816385) described in Example 1 at concentrations of 1.8, 0.6, or 0.2 μmol / kg. Tissues were collected 72 hours after the final injection to assess compound delivery and efficacy.

[0711] MALAT1 expression was detected using QuantiGene ViewRNA tissue assay (Affymetrix, catalog number QVT0011). Species-specific MALAT1 probes were purchased from Affymetrix (catalog number VB-11110-01 / mouse; VF1-13963 / monkey). In brief, mouse tissues were fixed in 10% neutral buffered formalin and embedded in paraffin and cut into 4-mm slices. After deparaffinization, tissue slides were boiled in Affymetrix pretreatment solution for 10-30 minutes, followed by protease treatment at 40°C for 10 to 40 minutes depending on the tissue. MALAT1 RNA probes were used at a 1:40 dilution and incubated with samples at 40°C for 120 minutes. After washing, the MALAT1 RNA / probe complex was hybridized with the preamplifier, amplifier, and AP-oligonucleotide at 40°C for 25, 15, and 15 minutes, respectively. After removing free AP oligonucleotides by washing in PBS, the slides were incubated with Fast Red substrate at room temperature for 30 minutes. Tissue images were acquired using an Aperio scanner. Hung et al., 2013 NucAcid Ther. [Nucleic Acid Therapeutics] 369-78.

[0712] In situ hybridization analysis indicates that GLP-1 peptide conjugation reduces MALAT1 staining of pancreatic β islet cells rather than acinar cells. ASO staining of pancreatic sections demonstrates that GLP-1 conjugates improve efficacy by increasing ASO delivery to tissues. Mice treated with GLP-1-conjugated MALAT1 ASO (ISIS 816385), but not mice treated with unconjugated MALAT1 ASO (ISIS 556089), show reduced MALAT1 expression in pancreatic β islet cells. Mice treated with various doses of ISIS 816385 show reduced MALAT1 expression in pancreatic β islet cells. Mice treated with GLP-1-conjugated MALAT1 ASO (ISIS 816385), but not mice treated with unconjugated MALAT1 ASO (ISIS 556089), show ASO accumulation in pancreatic β islet cells. GLP-1 conjugated MALAT1 ASO (ISIS 816385) accumulated in the pancreatic β islet cells of treated mice in a dose-dependent manner.

[0713] Study 2

[0714] To determine the dose response of the GLP-1 conjugated MALAT1 ASO (ISIS 816385) described in Example 1 on pancreatic MALAT1 expression, male C57BL / 6 mice fed a chow diet received a single intravenous injection of ISIS 816385 or the above unconjugated MALAT1 ASO (ISIS 556089) at concentrations of 0.2, 0.06, and 0.02 μmol / kg.

[0715] MALAT1 expression was detected using the QuantiGene View RNA tissue assay described above.

[0716] In situ hybridization analysis indicated that GLP-1 peptide conjugation reduced MALAT1 staining in pancreatic beta islet cells at the 0.2 μmol / kg dose and the 0.06 μmol / kg dose.No observable effects of ISIS 816385 or ISIS 556089 were observed in the liver for any dose.

[0717] Example 7: Antisense inhibition of MALAT1 and FOXO1 by GLP-1 peptide-conjugated antisense oligonucleotides in HEK293 cells overexpressing human GLP-1 receptor

[0718] Antisense oligonucleotides designed to target MALAT1 and FOXO1 were conjugated to glucagon-like peptide 1 receptor peptide agonist (GLP-1 peptide) and tested for their effects on human target gene expression using a HEK293 cell line with stable constitutive expression of the human GLP-1 receptor (hGLP1R-HEK).

[0719] By using Flp-IN TMhGLP1R was expressed in 293 cells to generate hGLP1R-HEK cell lines. Cultured hGLP1R-HEK cells were seeded in 96-well plates at a density of 30,000 cells per well and treated with saline, 100 nM or 10 μM unconjugated parent antisense oligonucleotides ISIS 556089 (targeting MALAT1 above) or ISIS 776102 (nucleobase sequence: TCTTCTTAAAATACCC) (SEQ ID NO: 64) (targeting FOXO1) or corresponding GLP-1 peptide-conjugated antisense oligonucleotides (ISIS 816385 targeting MALAT1 above or ION 913193 targeting FOXO1 above) for approximately 24 hrs. After the treatment period, cells were harvested, mRNA isolated, and adjusted to total RNA content as measured by a nanadrop UV-Vis spectrophotometer. MALAT1 or FOXO1 mRNA levels were measured by real-time quantitative PCR and normalized to the mRNA levels of the housekeeping gene (RPLP0) in the same sample. Human MALAT1 mRNA levels were measured using gene expression assay HS00273907, and FOXO1 mRNA levels were measured using assay Hs01054576 (Applied Biosystems). The mRNA levels of the housekeeping gene RPLP0 were measured using a primer probe set with forward sequence CCATTCTATCATCAACGGGTACAA (SEQ ID NO: 66), reverse sequence AGCAAGTGGGAAGGTGTAATCC (SEQ ID NO: 67).

[0720] Data are presented as the percentage inhibition of MALAT1 or FOXO1 mRNA relative to untreated control cells. Open symbols represent treatment with parent antisense oligonucleotides, while solid symbols represent treatment with antisense nucleotides conjugated to GLP-1 peptides. As the results illustrate, antisense oligonucleotides more effectively inhibit MALAT1 or FOXO1 mRNA in hGLP1R-HEK cell lines when conjugated to GLP-1 peptides compared to parent antisense oligonucleotides. The results show the percentage of FOXO1 mRNA and MALAT1 mRNA in HEK293 cells treated with unconjugated parent ASOs (ISIS 776102 or ISIS 556089) or GLP1-conjugated ASOs (ISIS 913193 or ISIS 816385) relative to antisense oligonucleotide (ASO) concentrations.

[0721] Example 8: Dose-dependent antisense inhibition of MALAT1 following treatment with unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides in wild-type and HEK293 cells overexpressing human GPR40 or GLP-1 receptors

[0722] The MALAT1 antisense oligonucleotides from Example 7 were further tested at various concentrations in wild-type, hGPR40 and hGLP1R-HEK cells.

[0723] Cultured hGLP1R-HEK, wild-type HEK293 (WT HEK293) cells or cells expressing hGPR40 receptors were seeded in 96-well plates at a density of 30,000 cells per well and treated with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 or 30 μM antisense oligonucleotides, concentrations as shown in the results, for about 24 hr. After the treatment period, cells were harvested, mRNA was isolated and MALAT1 mRNA levels were measured by real-time quantitative PCR using primer probe sets as described herein (Example 7). Data are expressed as MALAT1 mRNA levels normalized to housekeeping genes (RPLP0). Open symbols represent treatment with parental antisense oligonucleotides targeting MALAT1 (ISIS 556089), while solid symbols represent treatment with the same antisense nucleotides conjugated to GLP-1 peptides (ISIS 816385). Results show MALAT1 mRNA levels relative to antisense oligonucleotide (ASO) concentrations in HEK293 cells overexpressing the GLP1 receptor, wild-type HEK293 cells, or HEK293 cells overexpressing GRP40 treated with unconjugated parental MALAT1 ASO (ISIS 556089) or GLP1-conjugated MALAT1 ASO (ISIS 816385).

[0724] The half maximal inhibitory concentration (IC50) for each oligonucleotide is presented in the table below.

[0725] Table 1

[0726] treat hGLP1R-HEK cells WT HEK293 cells ISIS556089 IC50=0.87μM IC50=0.74μM ISIS816385 IC50=0.02μM IC50=2.21μM

[0727] The antisense oligonucleotides were 40-fold more potent in inhibiting MALAT1 gene expression when conjugated to a GLP-1 peptide agonist in the hGLP1R-HEK cell line but not in the WT HEK293 or hGPR40-HEK cell lines.

[0728] Example 9: Antisense inhibition of MALAT1 and FOXO1 in primary islets of Langerhans mice following treatment with unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides

[0729] The ability of antisense oligonucleotides targeting MALAT1 and FOXO1 to reduce gene expression was further tested in primary islets of Langerhans mice.

[0730] Islets were isolated by collagenase digestion of pancreases collected from 12-15 week old female C57BL / 6Crl mice that were bled to death. Islets were maintained in tissue culture until use. Islets were dissociated into single cells by shaking in a medium containing low extracellular calcium concentration. 10 to 20 intact or dissociated islets were plated on plastic culture dishes and treated with 10 μM antisense oligonucleotides for approximately 24 hr. After the treatment period, cells were harvested, RNA was isolated, and adjusted to total RNA content, such as by Measured. MALAT1 or FOXO1 mRNA levels were measured by real-time quantitative PCR. Mouse Malat1 mRNA levels were measured using the gene expression assay Mm01227912_s1 from Applied Biosystems, while mouse FOXO1 mRNA levels were measured using a primer probe set with forward sequence CAGTCACATACGGCCAATCC (SEQ ID NO: 68), reverse sequence CGTAACTTGATTTGCTGTCCTGAA (SEQ ID NO: 69), and probe sequence TGAGCCCTTTGCCCCAGATGCCTAT (SEQ ID NO: 70). All data were normalized to the mRNA level of a housekeeping gene (RPLP0) in the same sample, measured using a primer probe set with forward sequence GAGGAATCAGATGAGGATATGGGA (SEQ ID NO: 71), reverse sequence AAGCAGGCTGACTTGGTTGC (SEQ ID NO: 72), and probe sequence TCGGTCTCTTCGACTAATCCCGCCAA (SEQ ID NO: 73).

[0731] Data are shown as the levels of MALAT1 or FOXO1 mRNA relative to the housekeeping gene (RPLP0). Asterisks indicate no treatment, open circles indicate treatment with unconjugated antisense oligonucleotides (ISIS 556089 targeting MALAT1 or ISIS 776102 targeting FOXO1), open squares indicate treatment with scrambled FOXO1 antisense oligonucleotide sequences conjugated to GLP1 peptides (ION913195), and filled symbols indicate treatment with GLP1 peptide-conjugated antisense oligonucleotides against MALAT1 (ISIS 816385) or FOXO1 (ISIS 919193). The results show MALAT1 mRNA levels in dispersed mouse islets treated with no ASO, unconjugated parental MALAT1 ASO (ISIS556089), or GLP1-conjugated MALAT1 ASO (ISIS 816385); MALAT1 mRNA levels in intact mouse islets treated with no ASO, unconjugated parental MALAT1 ASO (ISIS 556089), or GLP1-conjugated MALAT1 ASO (ISIS 816385); and FOXO1 mRNA levels in intact mouse islets treated with no ASO, unconjugated parental FOXO1 ASO (ISIS 776102), GLP1-conjugated scrambled FOXO1 ASO (ION913195), or GLP1-conjugated FOXO1 ASO (ION 913193).

[0732] Example 10: Antisense inhibition of FOXO1 and reduction of Foxo1 protein in primary islets of Langerhans mice following treatment with unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides

[0733] Antisense oligonucleotides targeting FOXO1 were tested for their ability to reduce protein levels in primary islets of Langerhans mice.

[0734] Islets were isolated by collagenase digestion of pancreases collected from euthanized 12 to 15 week old female B6.Cg-Lepob / J mice and maintained in tissue culture until use. 150 complete islets were placed in plastic culture dishes, treated with 1 μM antisense oligonucleotides for 3 hr every 24 hr, and harvested after a total treatment time of approximately 24 hr, 48 hr or 96 hr, respectively. After the treatment period, islets were harvested, and half an islet was used to measure FOXO1 mRNA levels as described herein (Example 9). Half an islet was homogenized in an M-PER protein extraction reagent (Thermo Scientific) containing a protease inhibitor mixture (Complete Mini and phosphoSTOP, Roche Diagnostics). The protein content of the lysate was quantitatively determined using BCA assay reagent (Pierce). FoxO1 protein was detected by Western blot analysis using the primary antibody C29H4 (Cell Signalling, #2880) against FoxO1. α-Tubulin was measured using the primary antibody from Sigma (#T6074) as a control for sample loading on the gel. For the anti-FoxO1 antibody, the secondary antibody was HRP-conjugated polyclonal goat anti-rabbit P0448 (DAKO), and for the anti-α-tubulin antibody, the secondary antibody was HRP-conjugated polyclonal goat anti-mouse P0447 (DAKO). Enhanced chemiluminescence reagent (Pierce) was used for detection.

[0735] Inhibition of FOXO1 mRNA is presented as FOXO1 mRNA relative to housekeeping genes, expressed as a percentage of untreated cells in the table below, and shows a marginal reduction in mRNA for the unconjugated antisense oligonucleotide (ISIS 776102) and a reduction of more than 70% for the GLP-1 conjugated antisense oligonucleotide (ION 913193).

[0736] Table 2 Pancreatic islet FOXO1 mRNA levels relative to control (RPLP0)

[0737] treat 24 hours 48 hours 96 hours Carrier 100％ 100％ 100％ ISIS776102 82％ 86％ 92％ ION913193 31％ 30％ 25％

[0738] Western blot shows reduced FoxO1 protein levels measured in islets treated with vehicle or antisense oligonucleotides for 96 hours. Protein levels were quantified by measuring the intensity of the bands on the gel, normalized to the intensity of α-tubulin, and expressed as a percentage of vehicle-treated islets. FoxO1 protein levels in vehicle-treated islets were set at 100%. In contrast, FoxO1 protein levels were 5% in GLP-1-FOXO1 ASO-treated islets.

[0739] Example 11: In situ uptake of antisense oligonucleotides in pancreatic islets of Langerhans following administration of unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 to C57BL / 6Crl mice

[0740] Unconjugated parental and MALAT1-targeted GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the uptake of antisense oligonucleotides in islets following treatment with intravenous or subcutaneous administration.

[0741] Female C57BL / 6Crl mice were assigned to five treatment groups. Two groups received vehicle (saline) or 2 μmol / kg GLP-1 conjugated antisense oligonucleotides (ISIS 816385) by tail vein injection. Three groups received saline, 2 μmol / kg unconjugated parent antisense oligonucleotides (ISIS 556089) or 2 μmol / kg GLP-1 conjugated antisense oligonucleotides (ISIS 816385) twice a week by subcutaneous administration for two weeks. All animals were killed approximately 72hr after the last administration, and the pancreas was harvested for the uptake of antisense oligonucleotides analyzed in vitro by immunohistochemistry.

[0742] All tissues were fixed in 10% neutral buffered formalin for 32 h at room temperature. After fixation, samples were dehydrated with a standard ethanol series and subsequently dehydrated with xylene and embedded in paraffin. Tissue sections were cut at 4 μm thickness and mounted in Plus slides were then baked in a drying oven at 60°C for 1 hour. Immunohistochemistry for detecting antisense oligonucleotides was performed in a Ventana Discovery XT automatic stainer (Ventana Medical System, Inc) according to the manufacturer's recommendations, and all reagents were Ventana products (Roche Diagnostics, Basel, Switzerland). Protease 1 was used as an enzyme antigen repair and incubation lasted for 8 minutes. Antibody blocking agent was added for 4 minutes to reduce background, followed by rabbit anti-ASO 2.5 at 37°C for 1 hour (dilution 1:5000, Ionis Pharmaceuticals). For the second detection, OmiMap anti-rabbit HRP was incubated for 16 minutes, followed by chromogenic detection with DISCOVERY ChromoMap DAB kit (RUO). The slides were counterstained with hematoxylin for 4 minutes, followed by blueing for 4 minutes. The stained slides were analyzed under a standard bright field microscope.

[0743] The antisense oligonucleotides were detected in the islets of Langerhans of animals treated with ISIS 816385, which was administered either subcutaneously or intravenously. In animals treated subcutaneously with ISIS 556089, the antisense oligonucleotides were not detected in the islets of Langerhans.

[0744] Example 12: In situ antisense inhibition of MALAT1 in pancreatic islets of Langerhans following administration of unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice

[0745] Unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo against MALAT1 to evaluate antisense inhibition of MALAT1 in the pancreas following intravenous or subcutaneous treatment.

[0746] Female C57BL / 6Crl mice were assigned to five treatment groups as described herein (Example 11). All animals were sacrificed approximately 72 hr after the last dose and pancreases were harvested by in situ hybridization for ex vivo analysis of MALAT1 expression.

[0747] Tissues were prepared as described in Example 11 herein. VS assay, in situ mRNA amplification and labeling process was performed on Ventana Discovery ULTRA, an automated ISH platform (Ventana Medical System, Inc). Custom probes were obtained from ACD for detection of MALAT1 mRNA, and various parameters were tested to optimize the new RNAscope method for ISH. Multiple steps were used to amplify the signal, followed by labeled probes and using 2.5VS Reagent Kit-RED was used for detection. Stained slides were analyzed under a standard bright field microscope.

[0748] MALAT1 expression was reduced in the islets of Langerhans but not in the exocrine tissues of animals treated subcutaneously or intravenously with antisense oligonucleotides conjugated to GLP-1 peptide (ISIS816385). MALAT1 expression was not reduced in animals treated subcutaneously with the unconjugated parent antisense oligonucleotide (ISIS 556089).

[0749] Example 13: Uptake of antisense oligonucleotides in the liver 72 hours after administration of unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 to C57BL / 6Crl mice

[0750] Unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo against MALAT1 to assess the uptake of antisense oligonucleotides in the liver via intravenous or subcutaneous administration routes.

[0751] Animals were assigned to treatments as described herein in Example 11. All animals were sacrificed approximately 72 hr after the last dose and livers were harvested for analysis of antisense oligonucleotide uptake ex vivo by immunohistochemistry.

[0752] Tissues were prepared and immunohistochemistry performed as described in Example 12 herein.

[0753] Antisense oligonucleotides were detected in hepatocytes and Kupffer cells in the liver of animals treated with ISIS 816385 and ISIS 556089, which were administered by subcutaneous or intravenous administration as indicated. Example 14: Antisense inhibition of MALAT1 in the liver following administration of unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice

[0754] Unconjugated parent (ISIS556089) and GLP-1 peptide conjugated antisense oligonucleotides were further tested in vivo against MALAT1 by intravenous and subcutaneous administration routes to evaluate antisense inhibition of MALAT1 in the liver.

[0755] Female C57BL / 6Crl mice were assigned to five treatment groups as described herein (Example 13). All animals were sacrificed approximately 72 hr after the last treatment and livers were harvested for ex vivo analysis of MALAT1 expression by in situ hybridization.

[0756] Tissues were prepared and in situ hybridization was performed as described in Example 12 herein.

[0757] Hepatic MALAT1 expression was reduced to a greater extent in hepatocytes of animals treated with ISIS 816385 than in hepatocytes of animals treated with subcutaneous administration of ISIS 556089. Hepatic MALAT1 was also reduced compared to vehicle controls in animals administered ISIS 816385 by intravenous administration. Example 15: Dose-dependent antisense inhibition of MALAT1 in isolated islets of Langerhans and liver 72 hrs after administration of a single dose of unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice

[0758] Unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the efficacy of antisense inhibition of MALAT1 in isolated islets of Langerhans relative to liver 72 hours after a single subcutaneous administration.

[0759] Female C57BL / 6Crl mice are distributed into eight treatment groups, receive single subcutaneous injection vehicle, 0.01 μ mol / kg, 0.03 μ mol / kg, 0.1 μ mol / kg or 1 μ mol / kg ISIS 816385, and other three treatment groups receive 0.01 μ mol / kg, 0.1 μ mol / kg or 1 μ mol / kg ISIS 556089. All animals are put to death 72hr after the last administration. (Example 9) collects liver samples and separates islets as described herein, for mRNA analysis. (Example 9) quantitative MALAT1mRNA level as described herein, and is expressed as the percentage of vehicle-treated animals (control).

[0760] No significant antisense inhibition of MALAT1 was observed in the liver of any treatment group or in the islets of Langerhans of animals treated with the parent antisense oligonucleotide (ISIS 556089). GLP-1 peptide-conjugated antisense oligonucleotides dose-dependently inhibited MALAT1 mRNA levels with an estimated ED50 of 0.07 μmol / kg. Example 16: Dose-dependent antisense inhibition of FOXO1 in isolated islets of Langerhans and liver 72 hrs after administration of a single dose of unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice

[0761] Unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the efficacy of antisense inhibition of FOXO1 in isolated islets of Langerhans relative to the liver 72 hours after subcutaneous administration of a single dose.

[0762] Female C57BL / 6Crl mice were assigned to seven treatment groups, receiving a single subcutaneous injection of vehicle, 0.01 μmol / kg, 0.03 μmol / kg, 0.1 μmol / kg or 1 μmol / kg ION 913193, and two treatment groups received 0.01 μmol / kg or 1 μmol / kg ISIS 776102. All animals were sacrificed 72 hr after the last dose. Liver samples were collected and pancreatic islets were isolated as described herein (Example 9) for mRNA analysis. FOXO1 mRNA levels were quantified as described herein (Example 9) and expressed as a percentage of vehicle-treated animals (control).

[0763] No significant antisense inhibition of FOXO1 was observed in the liver of any treatment group or in the islets of Langerhans treated with the parent antisense oligonucleotide (ISIS 776102).GLP-1 peptide-conjugated antisense oligonucleotides dose-dependently inhibited FOXO1 mRNA levels with an estimated ED50 of 0.04 μmol / kg.

[0764] Example 17: Antisense Inhibition of FOXO1 in Isolated Islets of Langerhans and Liver Following Repeated Administration of Unconjugated Parental or GLP-1 Peptide-Conjugated Antisense Oligonucleotides for 6 Weeks in Ob / ob Mice

[0765] Unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the efficacy of antisense inhibition of FOXO1 in isolated islets of Langerhans versus liver after 6 weeks of treatment.

[0766] Male ob / ob mice (B6.V-Lepob / OlaHsd, Harlan) were assigned to five treatment groups, receiving vehicle, 0.1 μmol / kg ISIS 776102, 0.1 μmol / kg ION 913195, 0.03 ION 913193 or 1 μmol / kg ION913193. All animals were treated once a week for 6 weeks. Approximately 120 hr after the last dose, all animals were sacrificed, liver samples were collected and pancreatic islets were isolated as described herein (Example 9) for mRNA analysis. FOXO1 mRNA levels were quantified as described herein (Example 9), and the mRNA levels expressed relative to the housekeeping gene (RPLP0) in each sample were quantified.

[0767] No significant antisense inhibition of FOXO1 in the liver was observed in any treatment group or in islets of Langerhans treated with the parent antisense oligonucleotide (ISIS 776102) or with the scrambled FOXO1 antisense oligonucleotide sequence conjugated to the GLP-1 peptide (ION 913195). Animals treated with the GLP-1 peptide-conjugated antisense oligonucleotide (ION913193) had reduced FOXO1 mRNA levels in isolated islets of Langerhans at both dose levels tested (mean reduction of 42% in FOXO1 mRNA at 0.03 μmol / kg and 72% at 0.1 μmol / kg), indicating that GLP-1 peptide conjugation enhances antisense inhibition in islets of Langerhans in vivo.

[0768] Example 18: FoxO1 protein levels are reduced in islets of Langerhans isolated from ob / ob mice treated for 6 weeks with unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides

[0769] Unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides were further tested for their ability to reduce FoxO1 protein levels in islets of Langerhans isolated from ob / ob mice treated for 6 weeks.

[0770] Male ob / ob mice were assigned to five treatment groups as described herein (Example 17). Approximately 120 hr after the last dose, all animals were sacrificed and islets were isolated for FoxO1 protein analysis as described herein (Example 10). Random samples were selected from each treatment group and loaded onto each gel so that at least one sample from each treatment group was analyzed on the same gel. FoxO1 protein levels were measured by quantitative intensity and normalized to α-tubulin levels in the same sample. All samples within a single gel were expressed as a percentage of the levels measured in islets of animals receiving ION 913195.

[0771] Foxo1 protein levels were reduced in animals treated with ION 913193; 57% and 81% reductions relative to ION 913195 in animals receiving 0.03 μmol / kg and 0.1 μmol / kg, respectively, and 64% and 36% reductions in animals treated with 0.1 μmol / kg ISIS 776102.

[0772] Example 19: Preparation of GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1

[0773] A 5'-GLP-1 peptide-conjugated ASO targeting MALAT1 (ION 962963) was prepared according to the procedure of Example 1, starting with a 5'-hexylamino-modified oligonucleotide (ISIS 722061) (nucleobase sequence: GCATTCTAATAGCAGC (SEQ ID NO:65) with chemical modifications Gks mCks Aks Tds Tds mCds Tds Ads Ads Tds Ads Gds mCds AksGks mCk (k = cEt; d = 2'-deoxy; e = 2'-MOE; mC = 5-methylcytosine; o = phosphodiester; and s = phosphorothioate).

[0774] Example 20: Preparation of GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 via a click linker

[0775] Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position which is conjugated via a click linker.

[0776] Preparation of 5'-BCN MALAT-1 targeting oligonucleotide ISIS 791173:

[0777] A 5'-hexylamino modified oligonucleotide (ISIS786434) (nucleobase sequence: TCAGCATTCTAATAGCAGC (SEQ ID NO: 58) was synthesized and purified using a standard solid phase oligonucleotide procedure. The 5' end of the modified oligonucleotide contained a hexamethylene linker and a terminal amine. BCN-NHS ester (molecular weight 291.11 g / mol, 7441R, 8S, 9s)-bicyclo (6.1.0) non-4-yn-9-ylmethyl N-succinimidyl carbonate) was obtained from Aldrich. The modified oligonucleotide (approximately 1 g) was dissolved in 5 mL of sodium tetraborate buffer (pH 8.5). 13.4 mg of BCN-NHS ester was dissolved in 10 mL of DMSO, added to the ASO solution, and stirred at room temperature for 4 hours. The reaction mixture was diluted with 1 M NaCl solution and desalted by HPLC on a reverse phase column.

[0778] Preparation of GLP-1 click-conjugated ASO (ION 1071996)

[0779] 12mg modified oligonucleotide ISIS 791173 was dissolved in 1mL of 0.1M sodium tetraborate, pH 8.5 (ASO solution), and 12mg N-terminal azido-GLP-1 peptide was dissolved in 400μL DMF (peptide solution). The peptide solution was added to the ASO solution and stirred at room temperature for 18hr. At 18hr, a precipitate was observed and 1mL of additional DMF was added. The reaction was allowed to proceed for another 5hr. The product was purified by HPLC on a SAX column with buffer A 100mM NH4OAc / 30%ACN / H2O and buffer B 1.5M NaBr / NH4OAc / 30%ACN / H2O and desalted by HPLC on a reverse phase column. The product fractions were collected and cracked to yield the desired conjugated ASO, ION 1071996.

[0780]

[0781] Example 21: Antisense inhibition of MALAT1 in primary islets of Langerhans mice following treatment with unconjugated parent or GLP-1 peptide-conjugated ASOs with various linkers

[0782] To determine whether the conjugation chemistry of the GLP-1 peptide to the ASO affects the delivery of the ASO to the pancreas, male C57BL / 6 mice received intravenous injections of 0.6 μmol / kg / week of vehicle (saline), ISIS 556089 (parental unconjugated ASO), ISIS 816385 (GLP-1 conjugated ASO with a disulfide linker and a 5' TCA linker), ION 962963 (GLP-1 conjugated ASO with a disulfide linker and no 5' nucleotide spacer), or ION 1071996 (GLP-1 conjugated ASO conjugated via a click linker) once a week for three weeks. Tissues were collected 72 hours after the final injection to assess compound delivery and efficacy.

[0783] MALAT1 expression was detected as in Example 6. In situ hybridization analysis indicated that MALAT1 expression was reduced in beta islet cells in mice treated with GLP-1 conjugated ASOs (ISIS 816385, ION 962963, and ION 1071996) but not in mice treated with the unconjugated parental ASO (ISIS 556089) compared to saline controls.

[0784] Example 22: Dose-dependent reduction of MALAT-1 expression in LVX-GLP1R cells

[0785] HEK cells stably expressing FLAG-tagged GLP1R were generated by infecting HEK 293 cells with lentivirus containing FLAG-tagged GLP1R produced by transfecting 293T cells with pLVX-IRES-Puro (Clontech Laboratories Inc., Mountain View, CA) carrying the FLAG-GLP1R insert. Infected cells were selected with puromycin (2 μg / ml) and receptor expression was analyzed by Western blotting and immunofluorescence. Cultured GLP1R cells were plated at a density of 10,000 cells per well and treated with 0.3, 1, 3, 9, 27, 82, 247, 741, 2,222, 6,667, and 20,000 nM modified oligonucleotides for approximately 24 hours. After the treatment period, total RNA was prepared using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and qRT-PCR was performed using the primer probe set RTS2739 (forward sequence: AGGCGTTGTGCGTAGAGGAT (SEQ ID NO: 74), reverse sequence: AAAGGTTACCATAAGTAAGTTCCAGAAAA (SEQ ID NO: 75), probe sequence: AGTGGTTGGTAAAAATCCGTGAGGTCGGX (SEQ ID NO: 76)). Briefly, approximately 50 ng of total RNA in 5 μl of water was mixed with 0.3 μl of the primer probe set, which contained forward and reverse primers (10 μM each) and fluorescently labeled probe (3 μM) in a 20 μl reaction, 0.3 μl of RT enzyme mix (Qiagen), 4.4 μl of RNase-free water, and 10 μl of 2× PCR reaction buffer. Using StepOne Plus RT-PCR system (Applied Biosystems, Phoenix, Arizona, USA), reverse transcription was performed at 48°C for 10 min, 40 PCR cycles were performed at 94°C for 20 s, and 60°C for 20 s in each cycle. mRNA levels were determined by Ribogreen assay (Life Technologies) to determine the total RNA amount normalized to each reaction, and normalized to saline control (100% expression). The results are shown in the table below, and the dose-dependent inhibition of MALAT-1 and GLP-1 compounded ASOs (with TCA joints (816385) or without (962963)) was indicated.

[0786] Table 3: Percent inhibition of MALAT-1 expression in GLP1R HEK cells

[0787]

[0788]

[0789] Example 23: Effects of peptide length and conjugation position on the in vitro activity of GLP-1-conjugated ASOs targeting MALAT1

[0790] In order to assess the influence of precise peptide sequence, peptide length and conjugation position on the in vitro activity of the ASO conjugated to the GLP-1 complementary to MALAT1, a series of modified oligonucleotides with peptide sequence changes were synthesized via click chemistry. All peptides represent C-terminal amides. ION 1083582 was synthesized from ION 791173 via the click reaction of the lysine residue (X) modified with 5-azidopentanoic acid, as shown below. As shown in Example 20 above, other compounds were synthesized from ION 791173 via the click reaction with C-terminal azidonorleucine (Z).

[0791]

[0792] LVX-GLP1R cells (as described in Example 22) were plated at a density of 10,000 cells / well and incubated with 7 doses of peptide-conjugated ASO in a 4-fold dilution series. After the treatment period, total RNA was prepared and analyzed as described above in Example 22. The IC50s are shown in the table below.

[0793] Table 4: Effect of peptide composition and attachment point on activity of peptide-conjugated ASOs

[0794]

[0795] Example 24: Preparation of GLP-1-conjugated siRNA targeting PTEN

[0796] Method for preparing siRNA nucleotide duplexes targeting PTEN

[0797] ISIS 522247 (nucleobase sequence TTATCTATAATGATCAGGTAA (SEQ ID NO: 77) with chemical modifications Txs Ufs Amo Ufs Cmo Ufs AmoUfs Amo Afs Umo Gfs Amo Ufs Cms Afs Gms Gfs Ums Aes Ae (Tx = 5'-(E)-vinyl P-2'-O-methoxyethyl-thymine, f = 2'-α-fluoro-2'-deoxyribose, m = 2'-O-methyl ribose, e = 2'O-methoxyethyl ribose, o = phosphodiester; and s = phosphorothioate) and ISIS 790973 (nucleobase sequence ACCTGATCATTATAGATAA (SEQ ID NO: 78) with chemical modifications Afs Cms Cfo Umo Gfo Amo Ufo CmoAfo Umo Ufo Amo Ufo Amo Gfo Amo Ufs Ams Af (as above).

[0798] GLP-1 conjugated ION 1055394 was prepared according to the procedure of Example 1, starting with a 5'-hexylamino modified oligonucleotide (ION 1055395) (nucleobase sequence ACCTGATCATTATAGATAA (SEQ ID NO:78) with chemical modifications Afs CmsCfo Umo Gfo Amo Ufo Cmo Afo Umo Ufo Amo Ufo Amo Gfo Amo Ufs Ams Af (as above), conjugated to a GLP-1 peptide with the sequence HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleA laTrpLeuValLysGlyGlyProSerSerGlyAlaProProProSerCys comprising a free N-terminal amine and a C-terminal amide).

[0799] ISIS 522247 was hybridized with 790973 to produce a duplex of the two oligonucleotides. ISIS 522247 was hybridized with 1055394 to produce a duplex of the two oligonucleotides.

[0800] Example 25: Preparation of GLP-1 antagonist conjugated oligonucleotides

[0801] Methods for the synthesis of GLP-1 antagonist conjugated oligonucleotide (DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC-SS-Propionoyl-HA-o-Tdom CdoAdoGksmCksAks TdsTdsmCdsTdsAdsAdsTdsAdsGdsmCdsAksGksmCk (ION 998975)).

[0802] 38 mg of linker-ISIS 786434 (Compound 2) described in Example 1 was dissolved in 1.5 mL of H2O, and 0.5 mL of 0.1 M NaHCO3 / H2O was added to adjust the pH to about 7.5-8.0 (ASO solution).

[0803] 27.7 mg of peptide DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC-NH2 (SEQ ID NO: 79) was dissolved in 2 mL of DMF: 0.1 M NaHCO3 (1: 1) (peptide solution). The peptide solution was slowly added to the ASO solution and stirred at room temperature for 30 minutes. The reaction was monitored by LCMS and stirring was continued for another 1 hour. The major fraction was found to be the desired product. The product was diluted with water and kept at 4°C until it was purified by HPLC on a strong anion exchange column (buffer A = 100 mM ammonium acetate in 30% acetonitrile in water; buffer B: 28 column volumes, 1.5 M NaBr in A, 0 to 60% B). The fractions containing the full-length ASO were pooled together, diluted to give an acetonitrile concentration of 10%, and desalted by HPLC on a reverse phase column (buffer A 0.1 M sodium chloride, B = water, C = 50% acetonitrile in water). The fractions were pooled together and evaporated to yield the desired product confirmed by LCMS.

[0804] Example 26: Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated via a maleimide linker.

[0805] As described above, the 5' hexylamino modified oligonucleotide targeting MALAT1 (ISIS786434) was synthesized and purified. In sodium tetraborate buffer (pH 7), ISIS 786434 was reacted with 5 equivalents of N-succinimidyl 3-maleimido propionate (MW 266.21 g / mol) at room temperature to produce 5'-(3-maleimido) propionyl-C6MALAT1 ASO. The GLP-1 peptide containing C-terminal cysteine ​​amide ("GLP-1 peptide-cysteine ​​amide", HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2) was dissolved in 0.1M sodium phosphate, pH 8.5 / DMF, and added to 5'-(3-maleimido) propionyl-C6 MALAT1 ASO under stirring at room temperature. The product (ION 1086699) was formed.

[0806] Example 27: Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated via a disulfide click linker (preparation of Ionis-1123478).

[0807] 5'Hexylamino-modified oligonucleotide targeting MALAT1 (ISIS 786434) was used in AKTA Oligopilot TM On the synthesizer HL solid support (115mg, 47umol) was synthesized. Using standard solid phase oligonucleotide procedures, thiol modifier C6 SS amide (Glen Research 10-1936-02, 0.25g in 3.2525mL dry ACN) and N-(4-monomethoxytrityl (MMT))-6-amino-1-hexanolamide (0.177g in 3mL dry ACN) 0.1M solution was coupled to a solid support with Ion 925727. Cutting, precipitation and purification of 5'-MMT protected oligonucleotides as described above. The MMT protected oligonucleotides were dissolved in 50mL water and 50mL 3M sodium acetate solution (pH 5.0) and heated at 45°C for 60min. The reaction mixture was cooled to 22°C from 45°C and the pH was raised to 5.92 by adding 10% v / v 2.0M sodium acetate buffer solution (pH 7.2). The reaction was considered terminated when the addition of sodium acetate was complete. The product was purified by HPLC on source 30Q resin with buffer A 100 mM NH4OAc / 30% ACN / H2O and buffer B 100 mM NH4OAc / 30% ACN / H2O+1.5 M NaBr. Pure fractions were desalted by HPLC on a reverse phase column to yield MMT deprotected oligonucleotides.

[0808] The deprotected oligonucleotide was then reacted with 3 equivalents of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (744867 Aldrich) in 1:2 DMSO-sodium tetraborate buffer at pH 8.5 at room temperature to yield 5'-BCN-C6 MALAT1 ASO. The 5'-BCN-modified ASO was then desalted on a reverse phase column, dried and redissolved in 2 mL of sodium tetraborate buffer (pH 8.5). A GLP-1 peptide containing C-terminal 4-azidonorleucine ["GLP1 / Ex4 fusion sequence-40N3-NH2, H2N-HAibEGTFTSDVSSYLE EQAAKEFIAWLVKGGPSSGAPPPS(4-azidonorleucine)-NH2] was dissolved in 1 mL of DMSO and added to the ASO solution. After about 3 hr, the reaction mixture was diluted with water (5 times the volume of the reaction solution V / V) and the product was purified by HPLC on source 30Q resin with buffer A 100 mM NH4OAc / 30% ACN / H2O and buffer B 100 mM NH4OAc / 30% ACN / H2O+1.5 M NaBr. The product fractions were desalted by HPLC on reverse phase HPLC to yield ION 1123478 and confirmed by LC-MS analysis.

[0809]

[0810] Example 28: Method for preparing a conjugated modified oligonucleotide comprising GLP-1 at the 5' position conjugated via a maleimidic acid linker (preparation of Ionis-1123118).

[0811] Synthesize and purify the oligonucleotide (ISIS786434) of 5' hexylamino modification of targeting MALAT1 as described above.In sodium tetraborate buffer (pH 7), ISIS 786434 and 5 equivalents of N-succinimidyl 3-maleimido propionate (MW 266.21g / mol) are reacted at room temperature to produce 5'-(3-maleimido) propionyl-C6MALAT1 modified oligonucleotide.Use the linear gradient of buffer A and B, by SAX IE HPLC purification 5'-modified oligonucleotide.Buffer A: 100mM NH4OAc in 3:7 (v:v) acetonitrile: water, buffer B: 1.5M NaBr, 100mM NH4OAc in 3:7 (v:v) acetonitrile: water), and use reversed phase HPLC desalination. The GLP-1 peptide containing a C-terminal cysteine ​​amide ("GLP-1 peptide-cysteine ​​amide", HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2) was dissolved in 0.1M sodium phosphate, pH 7.0 / DMF and added to the 5'-(3-maleimido) propionyl-C6MALAT1 modified oligonucleotide under stirring at room temperature. The product (ION 1086699) was formed and purified by SAXIE HPLC using a linear gradient of buffers A and B. Buffer A: 50mM NaHCO3 in 3:7 (v:v) acetonitrile: water, buffer B: 1.5M NaBr, 50mM NaHCO3 in 3:7 (v:v) acetonitrile: water. The product fractions were pooled and kept at 5°C for 4 days and desalted by HPLC on a reverse phase column to yield ION 1123118 and confirmed by LC-MS analysis.

[0812]

[0813] Example 29: Antisense inhibition of MALAT1 in isolated islets of Langerhans 72 hrs after administration of a single dose of GLP-1 peptide-conjugated antisense oligonucleotides with different linkers in C57BL / 6Crl mice

[0814] To determine whether the conjugation chemistry of the GLP-1 peptide to the ASO affects the delivery of the ASO to the pancreas, female C57BL / 6Crl mice received a single subcutaneous injection of 0.01 μmol / kg of vehicle (saline), ISIS 816385 (GLP-1 conjugated ASO with a disulfide linker), ION 1071996 (GLP-1 conjugated ASO conjugated via a click linker), ION 1086699 (GLP-1 conjugated ASO conjugated via a maleimide linker), ION 1123118 (GLP-1 conjugated ASO conjugated via a maleimidic acid linker), or ION 1123478 (GLP-1 conjugated ASO conjugated via a disulfide click linker). Islets were isolated 72 hours after subcutaneous administration to assess the delivery and efficacy of the compounds.

[0815] MALAT1 expression was measured as in Example 9 and expressed as a percentage of vehicle-treated animals (control) and compared to expression in islets of mice treated with ISIS 816395. qPCR quantification showed that MALAT1 expression in beta islet cells was significantly further reduced in mice treated with GLP-1 conjugated ASOs ION 1086699, ION 1123118, and ION 1123478 compared to ISIS 816385, and was similarly reduced in mice treated with ION 1071996.

[0816]

[0817] Example 30: Dose-dependent antisense inhibition of MALAT1 in isolated islets of Langerhans 72 hrs after administration of a single dose of GLP-1 conjugated antisense oligonucleotides with different linkers in C57BL / 6Crl mice

[0818] To determine whether the conjugation chemistry of the GLP-1 peptide to the ASO affects the delivery of the ASO to the pancreas, female C57BL / 6Crl mice (n=6) received a single subcutaneous injection of ION 1086699 (GLP-1 conjugated MALAT-1 ASO conjugated via a maleimide linker), ION 1123118 (GLP-1 conjugated MALAT-1 ASO conjugated via a maleimidic acid linker), or ION 1134165 (GLP-1 conjugated MALAT-1 ASO conjugated via a disulfide linker) at 0.03, 0.01, 0.001, or 0.0003 μmol / kg. One group of mice (n=6) was subcutaneously injected with ISIS 816385 (GLP-1 conjugated MALAT-1 ASO conjugated via a disulfide linker as described in Example 1). A group of mice (n=6) were injected subcutaneously with PBS and used as a control group compared with other groups. Islets were isolated 72 hours after subcutaneous administration to evaluate the delivery and efficacy of the compound. The inhibition percentage of quantitative MALAT1 mRNA levels as described herein (Example 9) was taken for each treatment group with respect to the animals (control) treated with PBS. 0% inhibition reflects that the inhibition of MALAT1 mRNA was not observed. ND means that the specified dose was not applied to mice.

[0819] Table 6: Dose-Response Inhibition of MALAT-1 mRNA in Pancreatic Islets

[0820]

[0821] The results showed that GLP-1 conjugated MALAT-1 ASO conjugated via a maleimidic acid linker demonstrated greater dose-dependent inhibition of MALAT-1 mRNA in pancreatic islet cells compared to GLP-1 conjugated MALAT-1 ASO conjugated via a maleimide or disulfide linker.

[0822] Example 31: Effect of linkers on food intake in C57BL / 6Crl mice treated with GLP-1 conjugated antisense oligonucleotides

[0823] A single subcutaneous injection of 0.01, 0.03, or 0.1 μmol / kg of ION 816385 (MALAT-1 ASO conjugated to GLP-1 conjugated via a disulfide linker) or 0.1 μmol / kg of ION 1123118 (MALAT-1 ASO conjugated to GLP-1 conjugated via a maleimidic acid linker) was administered to a group of eight mice, as shown in the table below, and food intake was continuously measured for 24 hours. Food intake of eight mice administered PBS subcutaneous injection was monitored as a control. Compared with the disulfide linker, the maleimidic acid linker increases the therapeutic window in terms of the reduction in food intake induced by GLP1.

[0824] Table 7: Cumulative food intake (g)

[0825]

[0826]

Claims

1. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises: wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; n and o are independently selected from 2 to 10; and Y is attached directly or indirectly to the oligonucleotide.

2. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises: wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; n, o and p are independently selected from 2 to 10; m is 0 or 1; and Y is attached directly or indirectly to the oligonucleotide.

3. In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises: wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; m is 0 or 1; and Y is attached directly or indirectly to the oligonucleotide.

4. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises: wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; m is 1; and Y is attached directly or indirectly to the oligonucleotide.

5. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the compound comprises: wherein NN=N represents the azido group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remainder of the GLP-1 receptor ligand conjugate moiety; n and o are independently selected from 2 to 10; and Y is attached directly or indirectly to the oligonucleotide.

6. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: Where R=(CH2) n And n is from 1 to 12; X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

7. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: in and m is from 1 to 12; X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

8. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

9. A compound comprising an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

10. A composition comprising or consisting of a substantially pure mixture of two compounds, wherein The first compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: Where R=(CH2) n And n is from 1 to 12; X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety; and Y is directly or indirectly attached to said oligonucleotide; and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety, wherein the conjugate linker comprises: Where R=(CH2) n And n is from 1 to 12; X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

11. A composition comprising or consisting of a substantially pure mixture of two compounds, wherein The first compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: in and m is from 1 to 12; X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety; and Y is directly or indirectly attached to said oligonucleotide; and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: in and m is from 1 to 12; X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

12. A composition comprising or consisting of a substantially pure mixture of two compounds, wherein The first compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is directly or indirectly attached to said oligonucleotide; and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety via a conjugate linker, wherein the conjugate linker comprises: wherein X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

13. A compound as described in any one of claims 1-9 or a compositions as described in any one of claims 10-12, wherein the length of the oligonucleotide of the modification is 8 to 80 connected nucleosides.

14. A compound as described in any one of claims 1-9 or a compositions as described in any one of claims 10-12, wherein the length of the oligonucleotide of the modification is 10 to 30 connected nucleosides.

15. A compound as described in any one of claims 1-9 or a compositions as described in any one of claims 10-12, wherein the length of the oligonucleotide of the modification is 12 to 30 connected nucleosides.

16. A compound as described in any one of claims 1-9 or a compositions as described in any one of claims 10-12, wherein the length of the oligonucleotide of the modification is 15 to 30 connected nucleosides.

17. A compound or composition as claimed in any one of the preceding claims, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar, or at least one modified nucleobase.

18. The compound or composition of claim 17, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

19. compound or composition as claimed in claim 18, each modified internucleoside linkage of the oligonucleotide of wherein said modification is a phosphorothioate internucleoside linkage.

20. The compound or composition of any one of claims 17-19, wherein the modified sugar is a bicyclic sugar.

21. The compound or composition of claim 20, wherein the bicyclic sugar is selected from the group consisting of: 4'-(CH2)-O-2'(LNA); 4'-(CH2)2-O-2'(ENA); and 4'-CH(CH3)-O-2'(cEt).

22. The compound or composition of any one of claims 17-19, wherein the modified sugar is 2'-O-methoxyethyl, 2'-F, or 2'-OMe.

23. A compound or composition as described in any one of claims 17-22, wherein the modified nucleobase is 5-methylcytosine.

24. A compound or composition as claimed in any one of the preceding claims, wherein the modified oligonucleotide comprises: a gap segment consisting of linked deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides; wherein the gap segment is positioned adjacent to and between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.

25. A compound or composition as claimed in any one of the preceding claims, wherein the modified oligonucleotide is single-stranded.

26. The compound or composition of claim 25, wherein the modified oligonucleotide is an antisense oligonucleotide.

27. A compound or composition as described in claim 25, wherein the modified oligonucleotide is a miRNA antagonist or a miRNA mimic.

28. The compound or composition of any one of claims 1-24, wherein the compound comprises a double-stranded duplex.

29. The compound or composition of claim 28, wherein the double-stranded duplex comprises: a first strand of an oligonucleotide comprising said modification; and A second strand that is complementary to the first strand.

30. The compound or composition of claim 29, wherein the first strand of the oligonucleotide comprising the modification is complementary to an RNA transcript.

31. The compound or composition of claim 29, wherein the second strand is complementary to an RNA transcript.

32. The compound or composition of claim 28, wherein the compound is a miRNA mimic.

33. A compound or composition as described in any one of claims 1-32, wherein the compound comprises a ribonucleotide.

34. A compound or composition as described in any one of claims 1-32, wherein the compound comprises a deoxyribonucleotide.

35. A compound or composition as described in any one of claims 1-34, wherein the modified oligonucleotide is complementary to an RNA transcript in a cell.

36. The compound or composition of claim 35, wherein the cell is a pancreatic cell.

37. The compound or composition of claim 36, wherein the pancreatic cells are β-islet cells.

38. A compound or composition as described in any one of claims 35-37, wherein the RNA transcript is a pre-mRNA, mRNA, non-coding RNA, or miRNA.

39. The compound or composition of any one of claims 1-38, wherein the GLP-1 receptor ligand conjugate moiety is a peptide conjugate moiety, a small molecule conjugate moiety, an aptamer conjugate moiety, or an antibody conjugate moiety that targets the GLP-1 receptor.

40. The compound or composition of claim 39, wherein the peptide conjugate moiety is a GLP-1 peptide conjugate moiety.

41. A compound or composition as described in claim 40, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

42. A compound or composition as described in claim 41, wherein the GLP-1 peptide conjugate portion comprises conservative amino acid substitutions, amino acid analogs, or amino acid derivatives, wherein the conservative amino acid substitutions comprise replacing one aliphatic amino acid with another aliphatic amino acid; replacing serine with threonine, or replacing threonine with serine; replacing one acidic residue with another acidic residue; replacing one residue with an amide group with another residue with an amide group; exchanging one basic residue with another basic residue; or, replacing one aromatic residue with another aromatic residue, or a combination thereof, and the aliphatic residue comprises alanine, valine, leucine, isoleucine, or a synthetic equivalent thereof; the acidic residue comprises aspartic acid, glutamic acid, or a synthetic equivalent thereof; the residue comprising an amide group comprises aspartic acid, glutamic acid, or a synthetic equivalent thereof; the basic residue comprises lysine, arginine, or a synthetic equivalent thereof; or the aromatic residue comprises phenylalanine, tyrosine, or a synthetic equivalent thereof.

43. A compound or composition as described in claim 41, wherein the GLP-1 peptide conjugate portion comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the same length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

44. The compound or composition of claim 40, wherein the GLP-1 peptide conjugate portion is 8 to 50 amino acids in length and is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the amino acid sequence of any one of SEQ ID NOs: 1-57 over its entire length.

45. The compound or composition of claim 44, wherein the GLP-1 peptide conjugate moiety comprises conservative amino acid substitutions, amino acid analogs, or amino acid derivatives.

46. ​​The compound or composition of claim 44, wherein the entire length of the GLP-1 peptide conjugate portion is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-57.

47. The compound or composition of any one of claims 40-46, wherein the GLP-1 peptide conjugate moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-57.

48. The compound or composition of claim 47, wherein the GLP-1 peptide conjugate portion consists of the amino acid sequence of any one of SEQ ID NOs: 1-57.

49. The compound or composition of any one of claims 40-46, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 22), wherein Aib is aminoisobutyric acid.

50. The compound or composition of claim 49, wherein the GLP-1 peptide conjugate portion consists of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 22), wherein Aib is aminoisobutyric acid.

51. The compound or composition of any one of claims 40-46, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

52. The compound or composition of claim 51, wherein the GLP-1 peptide conjugate portion consists of the following amino acid sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Pen (SEQ ID NO: 23), wherein Aib is aminoisobutyric acid and Pen is penicillamine.

53. The compound or composition of any one of claims 40-46, wherein the GLP-1 peptide conjugate portion comprises the following amino acid sequence: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).

54. The compound or composition of claim 53, wherein the GLP-1 peptide conjugate portion consists of the following amino acid sequence: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).

55. The compound or composition of any one of claims 40-54, wherein the GLP-1 peptide conjugate moiety is capable of binding to a GLP-1 receptor.

56. The compound or composition of claim 55, wherein the GLP-1 receptor is expressed on the surface of a cell.

57. The compound or composition of claim 56, wherein the cell is a pancreatic cell.

58. The compound or composition of claim 57, wherein the pancreatic cells are β-islet cells.

59. A compound or composition as described in any one of claims 56-58, wherein the cell is in an animal.

60. A method of modulating expression of a nucleic acid target in a cell, the method comprising contacting the cell with a compound or composition as claimed in any one of the preceding claims, thereby modulating expression of the nucleic acid target in the cell.

61. The method of claim 60, wherein the cell is a pancreatic cell.

62. The method of claim 61, wherein the pancreatic cells are β-islet cells.

63. The method of any one of claims 60-62, wherein the cell expresses a GLP-1 receptor on the cell surface.

64. The method of any one of claims 60-63, wherein the cell is contacted with the compound to inhibit expression of the nucleic acid target.

65. The method of any one of claims 60 to 64, wherein: The nucleic acid target is a pre-mRNA, mRNA, non-coding RNA, or miRNA.

66. The method of any one of claims 60 to 65, wherein: The cell is within an animal.

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