GLP-1 receptor ligand moiety conjugated oligonucleotides and uses thereof

Abstract

To provide compounds and methods for targeting cells expressing GLP-1 receptor.SOLUTION: Disclosed is a compound comprising an oligonucleotide linked to GLP-1 receptor ligand conjugate moiety by a conjugate linker of a formula in which N-N=N is azido group of the GLP-1 receptor ligand conjugate moiety, and X directly or indirectly attaches to the remainder of the GLP-1 receptor ligand conjugate moiety; n and o are independently selected from 2-10; and Y attaches to the oligonucleotide directly or indirectly.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as a 29 kb file entitled BIOL0320USL2SEQ_ST25.txt, created on August 30, 2018. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0002] The present embodiments provide compounds and methods for targeting cells expressing the GLP-1 receptor. [Background technology]

[0003] The GLP-1 receptor is a class 2 G protein-coupled receptor that couples to adenylate cyclase via a stimulatory G protein receptor. Nutrient stimulation of the intestine releases glucagon-like peptide-1 into the circulation. Circulating GLP-1 binds to the GLP-1 receptor on the beta islet cells of the pancreas. This activates the GLP-1 receptor, inducing signaling events that result in insulin exocytosis from the beta islet cells. Binding of GLP-1 to the GLP-1 receptor results in the receptor being internalized into the cytoplasm and ultimately sorted into the lysosome (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kuna et al.,2013 Am J Physiol Endo Metab 305:E161-E170 Summary of the Invention [Means for solving the problem]

[0005] Embodiments provided herein relate to compounds and methods for modulating the expression of a nucleic acid target in a cell expressing a GLP-1 receptor. In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 receptor ligand conjugate moiety. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 receptor ligand conjugate moiety. In certain embodiments, contacting a cell expressing a GLP-1 receptor, such as a pancreatic beta islet cell, with a compound provided herein modulates the expression of a nucleic acid target in the cell. In certain embodiments, a compound comprising a GLP-1 receptor ligand conjugate moiety selectively or preferentially targets cells expressing a GLP-1 receptor compared to cells not expressing a GLP-1 receptor. In certain embodiments, a compound comprising a GLP-1 receptor ligand conjugate moiety selectively or preferentially targets cells expressing a GLP-1 receptor compared to a compound not comprising a GLP-1 receptor ligand conjugate moiety. [Brief explanation of the drawings]

[0006] [Figure 1] Graphs showing percent FOXO1 mRNA (Figure 1A) and MALAT1 mRNA (Figure 1B) versus antisense oligonucleotide (ASO) concentration in HEK293 cells treated with unconjugated parent ASO (ISIS 776102 or ISIS 556089) or GLP1-conjugated ASO (ISIS 913193 or ISIS 816385). [Figure 2] 2A and 2B are graphs showing MALAT1 mRNA levels versus antisense oligonucleotide (ASO) concentration in GLP1 receptor-overexpressing HEK293 cells (FIG. 2A), wild-type HEK293 cells (FIG. 2B), or GRP40-overexpressing HEK293 cells (FIG. 2C) treated with unconjugated parent MALAT1 ASO (ISIS 556089) or GLP1-conjugated MALAT1 ASO (ISIS 816385). [Figure 3]MALAT1 mRNA levels in dispersed mouse islet cells treated with no ASO, unconjugated parental MALAT1 ASO (ISIS 556089), or GLP1-conjugated MALAT1 ASO (ISIS 816385) (Figure 3A); MALAT1 mRNA levels in intact mouse islets treated with no ASO, unconjugated parental MALAT1 ASO (ISIS 556089), or GLP1-conjugated MALAT1 ASO (ISIS 816385) (Figure 3B); and FOXO1 mRNA levels in intact mouse islet cells treated with no ASO, unconjugated parental FOXO1 ASO (ISIS 776102), GLP1-conjugated scrambled FOXO1 ASO (ION 913195), or GLP1-conjugated FOXO1 ASO (ION 913193) (Figure 3C). Graph showing mRNA levels (FIG. 3C). DETAILED DESCRIPTION OF THE INVENTION

[0007] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed embodiments. As used herein, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "comprising" and other forms such as "includes" and "included" is not limiting.

[0008] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, papers, and GenBank and NCBI reference sequence records, are expressly incorporated herein by reference in their entirety and in part as discussed herein.

[0009] It is understood that the sequence set forth in each SEQ ID NO of the example oligonucleotides contained herein is independent of any modification to the sugar moiety, internucleoside linkage, or nucleobase. Thus, an oligonucleotide defined by a SEQ ID NO can independently contain one or more modifications to the sugar moiety, internucleoside linkage, or nucleobase. Oligonucleotides described by an ISIS or ION number (ISIS# or ION#) represent a combination of nucleobase sequence, chemical modification, and motif.

[0010] Throughout this specification, the first letter of a peptide sequence is understood to be the first amino acid of the peptide at the N-terminus, unless otherwise indicated, and the last letter of a peptide sequence is the last amino acid of the peptide at the C-terminus.

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

[0012] "2'-deoxynucleoside" means a nucleoside containing a 2'-H(H)furanosyl sugar moiety found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, 2'-deoxynucleosides may contain modified nucleobases or may contain RNA nucleobases (uracil).

[0013] "2'-O-Methoxyethyl" (also referred to as 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.

[0014] "2'-MOE nucleoside" (also referred to as 2'-O-methoxyethyl nucleoside) means a nucleoside that includes a 2'-MOE modified sugar moiety.

[0015] "2'-substituted nucleoside" or "2-modified nucleoside" means a nucleoside that includes a 2'-substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2-modified" with respect to a sugar moiety means a sugar moiety that includes at least one 2'-substituent other than H or OH.

[0016] "5-methylcytosine" means a cytosine having a methyl group attached to the 5 position.

[0017] "About" means within ±10% of a value. For example, if it is stated that "the compound caused 70% inhibition of the target nucleic acid," it means that the target nucleic acid level is inhibited within a range of 60% and 80%.

[0018] "Administration" or "administering" refers to the route by which a compound or composition provided herein is introduced into an individual to perform its intended function. One example of an administration route that can be used includes, but is not limited to, parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

[0019] "Aminoisobutyric acid" or "Aib", unless otherwise stated, has the formula: [ka] means 2-aminoisobutyric acid having the formula:

[0020] "Animal" refers to a human or non-human animal, including, but not limited to, a mouse, rat, rabbit, dog, cat, pig, and a non-human primate, including, but not limited to, a monkey and chimpanzee.

[0021] "Antisense activity" refers to any detectable and / or measurable activity resulting from 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 a target nucleic acid or a protein encoded by such a target nucleic acid compared to the target nucleic acid level or target protein level in the absence of an antisense compound directed against the target.

[0022] "Antisense compound" refers to a compound comprising an oligonucleotide and, optionally, one or more additional features, such as a conjugate group or a terminal group. Examples of antisense compounds include single-stranded and double-stranded compounds, such as oligonucleotides, ribozymes, siRNA, shRNA, ssRNA, and occupancy-based compounds.

[0023] "Antisense inhibition" means a reduction in target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

[0024] An "antisense mechanism" is any mechanism involving hybridization of a compound with a target nucleic acid where the outcome or effect of the hybridization is either target degradation or target occupancy with the concomitant stalling of cellular machinery, including, for example, transcription or splicing.

[0025] "Antisense oligonucleotide" means an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid, or a region or segment thereof. In certain embodiments, an antisense oligonucleotide is capable of specifically hybridizing to a target nucleic acid, or a region or segment thereof.

[0026] "Bicyclic nucleoside" or "BNA" refers to a nucleoside comprising a bicyclic sugar moiety. "Bicyclic sugar" or "bicyclic sugar moiety" refers to a modified sugar moiety comprising two rings, the second ring being 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.

[0027] "Branched group" means a group of atoms having at least three positions capable of forming covalent bonds to at least three groups. In certain embodiments, the branched group provides multiple reactive sites for linking a tethered ligand to an oligonucleotide via a conjugate linker and / or a cleavable moiety.

[0028] "Cell targeting moiety" means a conjugate group or a portion of a conjugate group that is capable of binding to one or more specific cell types.

[0029] "cEt" or "constrained ethyl" means a bicyclic furanosyl sugar moiety containing a bridge connecting the 4'-carbon and 2'-carbon, the bridge having the formula: 4'-CH(CH3)-O-2'.

[0030] A "chemical modification" in a compound describes the substitution or alteration of any of the units in that compound via a chemical reaction. A "modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and / or a modified nucleobase. A "modified oligonucleotide" means an oligonucleotide containing at least one modified internucleoside linkage, modified sugar, and / or modified nucleobase.

[0031] A "chemically distinct region" refers to a region of a compound that is chemically different in some sense from another region of the same compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides that do not have 2'-O-methoxyethyl modifications.

[0032] "Chimeric antisense compound" means an antisense compound having at least two chemically distinct regions, each position having multiple subunits.

[0033] "Cleavable bond" means any chemical bond that can be split. In certain embodiments, the cleavable bond is selected from among an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a disulfide, or a peptide.

[0034] "Cleavable moiety" means a bond or group that is cleaved under physiological conditions, eg, inside a cell, animal, or human.

[0035] "Complementary," with respect to oligonucleotides, means that the nucleobase sequence of such oligonucleotide, or one or more regions thereof, matches the nucleobase sequence of another oligonucleotide or nucleic acid, or one or more regions thereof, when the two nucleobase sequences are aligned in reverse orientation. Nucleobase matches or complementary nucleobases, as described herein, unless otherwise specified, include the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (C). m The term "fully complementary" or "100% complementary" refers to an oligonucleotide that has a nucleobase match at each nucleoside without any nucleobase mismatches.

[0036] "Conjugate group" means a group of atoms attached to an oligonucleotide. The conjugate group includes a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[0037] "Conjugate linker" means a group of atoms containing at least one bond that connects a conjugate moiety to an oligonucleotide.

[0038] "Conjugate moiety" means a group of atoms attached to an oligonucleotide via a conjugate linker.

[0039] "Designing" or "designed to" refers to the process of designing a compound that specifically hybridizes with a selected nucleic acid molecule.

[0040] "Differentially modified" refers to chemical modifications or chemical substituents that differ from one another, including the absence of modifications. Thus, for example, an MOE nucleoside and an unmodified DNA nucleoside are "differentially modified," even if the DNA nucleoside is unmodified. Similarly, DNA and RNA are "differentially modified," even if both are naturally occurring, unmodified nucleosides. Nucleosides that are identical except for containing different nucleobases are not differentially modified. For example, a nucleoside containing a 2'-OMe-modified sugar and an unmodified adenine nucleobase is not differentially modified, as is a nucleoside containing a 2'-OMe-modified sugar and an unmodified thymine nucleobase.

[0041] "Double-stranded antisense compound" means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, wherein one of the two oligomeric compounds comprises an oligonucleotide.

[0042] "Expression" includes all functions by which a gene's coded information is converted into structures present and operating in a cell, including, but not limited to, the products of transcription and translation.

[0043] "Gapmer" means an oligonucleotide comprising an internal region having multiple nucleosides that support RNase H cleavage located between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleosides or nucleotides comprising the external regions. The internal region may be referred to as the "gap," and the external regions may be referred to as the "wings."

[0044] "Hybridization" refers to the annealing of oligonucleotides and / or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleic acid bases. 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.

[0045] "Inhibiting expression or activity" refers to a reduction or disruption of expression or activity relative to expression of the activity in an untreated or control sample, and does not necessarily indicate a complete elimination of expression or activity.

[0046] "Internucleotide linkage" means a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. "Modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring phosphate internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages.

[0047] "Linked nucleosides" means adjacent nucleosides that are linked together by an internucleoside bond.

[0048] "Linker-nucleoside" refers to a nucleoside that connects an oligonucleotide to a conjugate moiety. The linker-nucleoside is located within the conjugate linker of the compound. The linker-nucleoside is not considered part of the oligonucleotide portion of the compound, even if it is contiguous with the oligonucleotide.

[0049] "Mismatch" or "non-complementary" refers to a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of the second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned. For example, a nucleobase, including but not limited to the universal nucleobases inosine and hypoxanthine, can hybridize with at least one nucleobase, but is still mismatched or non-complementary to the nucleobase to which it hybridizes. As another example, when the first and second oligonucleotides are aligned, a nucleobase of a first oligonucleotide that cannot hybridize with the corresponding nucleobase of the second oligonucleotide or target nucleic acid is a mismatched or non-complementary nucleobase.

[0050] "Modulating" refers to changing or adjusting a characteristic in a cell, tissue, organ, or organism. For example, modulating a target nucleic acid can mean increasing or decreasing the level of the target nucleic acid in a cell, tissue, organ, or organism. A "modulator" brings about a change in a cell, tissue, organ, or organism. For example, a compound can be a modulator that decreases the amount of a target nucleic acid in a cell, tissue, organ, or organism.

[0051] "MOE" means methoxyethyl.

[0052] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides.

[0053] "Motif" means the pattern of unmodified and / or modified sugar moieties, nucleobases and / or internucleoside linkages in an oligonucleotide.

[0054] "Natural" or "naturally occurring" means something found in nature.

[0055] "Non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" means a modified sugar moiety that contains a modification, such as a substituent, that does not bridge 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 acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

[0056] "Nucleobase" refers to a heterocyclic moiety that can pair with a base of another nucleic acid. As used herein, "naturally occurring nucleobases" are adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). A "modified nucleobase" is a naturally occurring nucleobase that has been chemically modified. A "universal base" or "universal nucleobase" is a nucleobase other than naturally occurring and modified nucleobases, and can pair with any nucleobase.

[0057] "Nucleobase sequence" means the order of consecutive nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage.

[0058] "Nucleoside" refers to a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each independently unmodified or modified. "Modified nucleoside" refers to a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.

[0059] "Oligomeric compound" means a compound comprising a single oligonucleotide and optionally one or more additional features, such as a conjugate group or a terminal group.

[0060] "Oligonucleotide" refers to a polymer of linked nucleosides, each of which may be modified or unmodified independently of one another. Unless otherwise specified, an oligonucleotide consists of 8 to 80 linked nucleosides. "Modified oligonucleotide" refers to an oligonucleotide in which at least one sugar, nucleobase, or internucleoside linkage is modified. "Unmodified oligonucleotide" refers to an oligonucleotide that does not contain any sugar, nucleobase, or internucleoside modifications.

[0061] "Parent oligonucleotide" means an oligonucleotide whose sequence is used as a basis for the design of more oligonucleotides of similar sequence but for different lengths, motifs, and / or chemical structures. The newly designed oligonucleotides may have sequences identical to or overlapping with the parent oligonucleotide.

[0062] "Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.

[0063] "Phosphorus moiety" means a group of atoms that includes a phosphorus atom. In certain embodiments, the phosphorus moiety includes a mono-, di-, or triphosphate or a phosphorothioate.

[0064] "Portion" means a defined number of consecutive (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of consecutive nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of consecutive nucleobases of an oligomeric compound.

[0065] "Reduce" means to make smaller in area, size, amount, or number.

[0066] "RNAi compound" means an antisense compound that acts, at least in part, through RISC or Ago2, but not 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.

[0067] A "segment" is defined as a smaller or subportion of a region within a nucleic acid.

[0068] "Selective" with respect to an effect refers to an effect on one that is greater than another by any quantitative range or fold difference. For example, a compound that includes a GLP-1 receptor-conjugated ligand moiety that is "selective" for cells expressing GLP-1 receptors, or that "selectively" targets cells expressing GLP-1 receptors, will target cells expressing GLP-1 receptors to a greater extent than a compound that does not include a GLP-1 receptor-conjugated ligand moiety. As another example, a compound that includes a GLP-1 receptor-conjugated ligand moiety that is "selective" for cells expressing GLP-1 receptors, or that "selectively" targets cells expressing GLP-1 receptors, will target cells expressing GLP-1 receptors to a greater extent than cells that do not express GLP-1 receptors or that express them at relatively low levels. It will be understood that the term "selective" does not require absolute all-or-none selectivity.

[0069] "Single-stranded" in reference to a compound means that the compound has only one oligonucleotide. "Self-complementary" means that the oligonucleotide is at least partially hybridized to itself. A compound that consists of one oligonucleotide and that is self-complementary is a single-stranded compound. A single-stranded compound can bind to a complementary compound to form a double-stranded compound.

[0070] A "site" is defined as a unique nucleobase position within a target nucleic acid.

[0071] "Specifically hybridizable" refers to an oligonucleotide having a degree of complementarity between the oligonucleotide and the target nucleic acid sufficient to induce a desired effect while exhibiting minimal or no effect on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.

[0072] "Specifically inhibit" with reference to a target nucleic acid means reducing or blocking expression of the target nucleic acid with lesser or minimal effect or no effect at all on non-target nucleic acids. Reduction does not necessarily indicate complete elimination of expression of the target nucleic acid.

[0073] "Standard cell assay" means the assay described in the Examples and reasonable variations thereof.

[0074] "Standard in vivo experiments" means the procedures described in the Examples and reasonable variations thereof.

[0075] "Sugar moiety" refers to an unmodified sugar moiety or a modified sugar moiety. "Unmodified sugar moiety" or "unmodified sugar" refers to a 2'-OH(H) furanosyl moiety found in RNA (an "unmodified RNA sugar moiety") or a 2'-H(H) moiety found in DNA (an "unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen at each of the 1', 3', and 4' positions, an oxygen at the 3' position, and two hydrogens at the 5' position. "Modified sugar moiety" or "modified sugar" refers to a modified furanosyl sugar moiety or sugar surrogate. "Modified furanosyl sugar moiety" refers to a furanosyl sugar containing a non-hydrogen substituent in place of at least one hydrogen of the unmodified sugar moiety. In certain embodiments, the modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic and non-bicyclic sugars.

[0076] "Sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can attach a nucleobase to another group within an oligonucleotide, such as an internucleoside linkage, a conjugate group, or a terminal group. Modified nucleosides containing sugar surrogates can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary compounds or nucleic acids.

[0077] "Target gene" refers to a gene that encodes a target.

[0078] "Targeting" in reference to a target nucleic acid refers to specific hybridization of an oligonucleotide to said target nucleic acid, which induces a desired effect. "Targeting" in reference to the GLP-1 receptor refers to the binding of a GLP-1 receptor ligand conjugate moiety to the GLP-1 receptor.

[0079] "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.

[0080] "Target region" means a portion of a target nucleic acid to which one or more compounds are targeted.

[0081] "Target segment" means the sequence of nucleotides of a target nucleic acid to which a compound is targeted. "5' target site" refers to the 5'-most nucleotide of a target segment. "3' target site" refers to the 3'-most nucleotide of a target segment.

[0082] "Terminal group" means a chemical group or group of atoms covalently attached to the terminus of an oligonucleotide.

[0083] Specific Embodiments In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 receptor ligand conjugate moiety. 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 moiety.

[0084] 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.

[0085] In certain embodiments, the oligonucleotide is a modified oligonucleotide comprising at least one modified internucleoside linkage, at least one modified sugar, or at least one modified nucleobase. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, each modified internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0086] 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.

[0087] In certain embodiments, the modified nucleobase is 5-methylcytosine.

[0088] In certain embodiments, 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 immediately adjacent to and between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar.

[0089] In certain embodiments, the oligonucleotide is single-stranded.

[0090] In certain embodiments, the oligonucleotide is an antisense oligonucleotide, an miRNA antagonist, or an miRNA mimic.

[0091] In certain embodiments, the compound comprises a duplex. In certain embodiments, the duplex comprises a first strand comprising a modified oligonucleotide and a second strand complementary to the first strand. In certain embodiments, the first strand comprising the modified oligonucleotide is complementary to an RNA transcript. In certain embodiments, the second strand is complementary to the RNA transcript. In certain embodiments, the compound comprises a 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 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 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.

[0092] In certain embodiments, the compound is an miRNA mimic.

[0093] In certain embodiments, the compound comprises ribonucleotides. In certain embodiments, the compound comprises deoxyribonucleotides.

[0094] In certain embodiments, the oligonucleotide is complementary to an RNA transcript in a cell, such as a pancreatic cell or a pancreatic beta islet cell.

[0095] In certain embodiments, the RNA transcript is a pre-mRNA, an mRNA, a non-coding RNA, or an miRNA.

[0096] 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.

[0097] In certain embodiments, the peptide conjugate moiety is a GLP-1 peptide conjugate moiety.

[0098] In certain embodiments, the GLP-1 peptide conjugate moiety comprises at least an 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 portion that 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 an equal-length portion of the amino acid sequence of any of SEQ ID NOs: 1-57.

[0099] In certain embodiments, the GLP-1 peptide conjugate moiety comprises at least an 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 portion that 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 an equal-length portion of the amino acid sequence of any of SEQ ID NOs: 1-57.

[0100] In certain embodiments, the GLP-1 peptide conjugate moiety 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 over its entire length to the amino acid sequence of any of SEQ ID NOs: 1-57.

[0101] In certain embodiments, the GLP-1 peptide conjugate moiety 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 over its entire length to the amino acid sequence of any of SEQ ID NOs: 1-57.

[0102] In certain embodiments, the GLP-1 peptide conjugate moiety is GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, which in the conventional three letter notation 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). 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 contiguous amino acids 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 an equal length portion of the amino acid sequence of

[0103] In certain embodiments, the GLP-1 peptide conjugate moiety comprises at least an 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 portion that 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 an isometric portion of the amino acid sequence of GLP-1(7-37).

[0104] In certain embodiments, the GLP-1 peptide conjugate moiety 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 over its entire length to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0105] In certain embodiments, the GLP-1 peptide conjugate moiety comprises a conservative amino acid substitution, an amino acid analog, or an amino acid derivative.

[0106] In certain embodiments, the GLP-1 peptide conjugate moiety 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 over its entire length to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0107] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0108] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0109] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (which in the conventional three letter notation 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)).

[0110] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the amino acid sequence of GLP-1(7-36)amide: (which, in the conventional three letter notation, 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)).

[0111] In certain embodiments, the GLP-1 peptide conjugate moiety comprises or consists of the amino acid sequence of GLP-1(7-36):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (which in the conventional three letter notation 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)).

[0112] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in the conventional three letter notation 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)).

[0113] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in the conventional three letter notation 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)).

[0114] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG (which in the conventional three letter code 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)).

[0115] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG (which in the conventional three letter code 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)).

[0116] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the amino acid sequence of any of SEQ ID NOs: 1-57.

[0117] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the amino acid sequence of any of SEQ ID NOs: 1-57.

[0118] In certain embodiments, the GLP-1 peptide conjugate moiety can be the C-terminal amide or acid of any of SEQ ID NOs: 1-57.

[0119] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the 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.

[0120] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the 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.

[0121] In certain embodiments, the GLP-1 peptide conjugate moiety comprises the 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.

[0122] In certain embodiments, the GLP-1 peptide conjugate moiety consists of the 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.

[0123] In certain embodiments, the GLP-1 peptide conjugate moiety is capable of binding to the GLP-1 receptor.

[0124] In certain embodiments, the GLP-1 receptor is expressed on the surface of the cell.

[0125] In certain embodiments, the cells are pancreatic cells, such as pancreatic beta islet cells.

[0126] In certain embodiments, the cell is in an animal.

[0127] 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.

[0128] In certain embodiments, the conjugate linker attaches the GLP-1 receptor ligand conjugate moiety to the 5' end of the oligonucleotide.

[0129] In certain embodiments, the conjugate linker attaches the GLP-1 receptor ligand conjugate moiety to the 3' end of the oligonucleotide.

[0130] In certain embodiments, the conjugate linker is cleavable.

[0131] In certain embodiments, the conjugate linker comprises a disulfide bond.

[0132] In certain embodiments, a disulfide bond links the GLP-1 peptide conjugate moiety to the oligonucleotide.

[0133] In certain embodiments, a disulfide bond joins the C-terminus of the GLP-1 peptide conjugate moiety to the 5' terminus of the oligonucleotide.

[0134] In certain embodiments, the conjugate linker comprises 1 to 5 linker-nucleosides.

[0135] In certain embodiments, the conjugate linker comprises three linker-nucleosides.

[0136] In certain embodiments, three linker-nucleosides have a TCA motif.

[0137] In certain embodiments, 1 to 5 linker-nucleosides do not contain a TCA motif.

[0138] In certain embodiments, the conjugate linker comprises a hexylamino group.

[0139] In certain embodiments, the conjugate linker comprises a polyethylene glycol group.

[0140] In certain embodiments, the conjugate linker comprises a triethylene glycol group.

[0141] In certain embodiments, the conjugate linker comprises a phosphate group.

[0142] In certain embodiments, the conjugate linker is [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly 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 by a disulfide bond.

[0143] In certain embodiments, the conjugate linker is [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety; and T1 comprises a modified oligonucleotide; and Bx is a modified or unmodified nucleobase. In certain embodiments, X comprises a disulfide bond.

[0144] In certain embodiments, the conjugate linker is [ka] (In the formula, the phosphate group is linked to the modified oligonucleotide and Y is linked to the conjugate group; Y is a phosphodiester or amino (—NH—) group; Z is a group of the formula: [ka] is a pyrrolidinyl group having the formula: j is 0 or 1; n is from about 1 to about 10; p is 1 to about 10; m is 0 or 1 to 4; and When Y is amino, m is 1. Includes.

[0145] In certain embodiments, Y is amino (—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 2 to 10 and p is 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 is a group of the formula: [ka] It has.

[0146] In certain embodiments, n is 2 and p is 3. In certain embodiments, n is 5 and p is 6.

[0147] In certain embodiments, the conjugate linker is [ka] Includes.

[0148] In certain embodiments, the conjugate linker is [ka] Includes.

[0149] In certain embodiments, the compound comprising the conjugate linker is [ka] (In the ceremony N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0150] In certain embodiments, the compound comprising the conjugate linker is [ka] (In the ceremony N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0151] In certain embodiments, the compound comprising the conjugate linker is [ka] (In the ceremony N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0152] In certain embodiments, the composition comprises at least one compound described herein. In certain embodiments, the pharmaceutical composition comprises at least one compound described herein and a pharmaceutically acceptable excipient.

[0153] In certain embodiments, a method for modulating expression of a target nucleic acid in a cell comprises contacting the cell with a compound of any of the preceding embodiments, thereby modulating expression of the nucleic acid target in the cell. In certain embodiments, the cell expresses a GLP-1 receptor on the surface of the cell. In certain embodiments, the cell is a pancreatic cell, such as a beta islet cell. In certain embodiments, the cell is a pituitary cell, a leptomeningeal 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 cardiac cell, a thyroid 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, the 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, the 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, nephroblastoma, 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, contacting a cell with a compound of any of the foregoing embodiments inhibits expression of a nucleic acid target. In certain embodiments, the nucleic acid target is a pre-mRNA, mRNA, non-coding RNA, or miRNA. In certain embodiments, the cell is in an animal.

[0154] In certain embodiments, a method for modulating expression of a target nucleic acid in an animal comprises administering to the animal a compound of any of the preceding embodiments, thereby modulating expression of the target nucleic acid in the animal. In certain embodiments, expression of the nucleic acid target is modulated in a cell of the animal that expresses a GLP-1 receptor on the surface of the cell. In certain embodiments, expression of the nucleic acid target is modulated in a pancreatic cell, such as a beta islet cell, of the animal. In certain embodiments, the cell is a pancreatic cell, such as a beta islet cell. In certain embodiments, the cell is a pituitary cell, a leptomeningeal cell, a duodenal cell, an ileal cell, a colonic 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, the 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, the 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, nephroblastoma, and neuroblastoma. In certain embodiments, the cancer is an ovarian cancer, prostate cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, lung cancer, and lymphoma. In certain embodiments, administering the compound inhibits 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.

[0155] Also provided herein is the use of a compound described herein for the manufacture of a medicament in the treatment of cancer. Also provided herein is a compound described herein for use in the treatment of cancer.

[0156] In certain embodiments, the method for preparing a compound comprises: [ka] wherein X1 is an oligonucleotide and the compound is a GLP-1 peptide-conjugated oligonucleotide. with a GLP-1 peptide.

[0157] In certain embodiments, the method for preparing a compound comprises: An oligonucleotide comprising a hexamethyl linker and a terminal amine at the 5' end of the oligonucleotide is prepared by reacting a nucleotide of the formula: [ka] with 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) having the formula: [ka] (wherein X1 is an oligonucleotide); and obtaining a compound 2 having the formula: Compound 2 is reacted with a GLP-1 peptide, thereby forming a compound of the formula: [ka] wherein X1 is an oligonucleotide and X2 is a GLP-1 peptide. and obtaining a GLP-1 peptide-conjugated oligonucleotide having Includes.

[0158] In certain embodiments, the method for preparing a GLP-1 peptide-conjugated oligonucleotide comprises: A solution containing an oligonucleotide comprising a hexamethyl linker and a terminal amine at the 5' end of the oligonucleotide is treated with a compound of the formula: [ka] with a solution containing 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) having the formula: [ka] (wherein X1 is an oligonucleotide) and obtaining a compound 2 having the formula: The solution containing Compound 2 is mixed with the solution containing the GLP-1 peptide, thereby forming a compound of the formula: [ka] wherein X1 is an oligonucleotide and X2 is a GLP-1 peptide. and obtaining a GLP-1 peptide-conjugated oligonucleotide having Includes.

[0159] In certain embodiments, the solution containing the oligonucleotide comprises a sodium phosphate buffer, and the solution containing 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) comprises dimethylformamide.

[0160] In certain embodiments, the solutions are mixed at room temperature.

[0161] In certain embodiments, the solution containing Compound 2 further comprises acetonitrile and NaHCO 3 and has a pH of about 8.0.

[0162] In certain embodiments, the solution comprising the GLP-1 peptide further comprises dimethylformamide.

[0163] In any of the aforementioned methods for preparing a compound or GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise at least an 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 contiguous amino acid portion that 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 an equal-length portion of the amino acid sequence of any of SEQ ID NOs: 1-57.

[0164] In any of the aforementioned methods for preparing a compound or GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may comprise at least an 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 contiguous amino acid portion that 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 an equal-length portion of the amino acid sequence of any of SEQ ID NOs: 1-57.

[0165] In any of the aforementioned 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 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 over its entire length to the amino acid sequence of any of SEQ ID NOs: 1-57.

[0166] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may 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 over its entire length to the amino acid sequence of any of SEQ ID NOs: 1-57.

[0167] In any of the foregoing methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may be GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which, in the conventional three letter notation, 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-Le) The amino acid sequence of the present invention 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 acids 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 an equal length portion of the amino acid sequence of u-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).

[0168] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise at least an 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 contiguous amino acid portion that 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 an equal-length portion of the amino acid sequence of GLP-1(7-37).

[0169] In any of the aforementioned 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 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 over its entire length to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0170] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may 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 over its entire length to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0171] In any of the foregoing methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0172] In any of the foregoing methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0173] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (which in the conventional three letter notation 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)).

[0174] In any of the foregoing methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the amino acid sequence of GLP-1(7-36)amide (SEQ ID NO:2).

[0175] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the amino acid sequence of GLP-1(7-36):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, which in the conventional three letter notation 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).

[0176] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG, which in the conventional three letter notation 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).

[0177] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in the conventional three letter notation 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)).

[0178] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the 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).

[0179] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the 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).

[0180] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the amino acid sequence of any of SEQ ID NOs: 1-57.

[0181] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the amino acid sequence of any of SEQ ID NOs: 1-57.

[0182] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the 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.

[0183] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the 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.

[0184] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can comprise the 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), where Aib is aminoisobutyric acid and Pen is penicillamine.

[0185] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can consist of the 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), where Aib is aminoisobutyric acid and Pen is penicillamine.

[0186] In any of the foregoing methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can contain a reactive sulfur moiety.

[0187] In any of the foregoing methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide can include penicillamine.

[0188] In any of the aforementioned methods of preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, penicillamine may be attached to the C-terminus of the GLP-1 peptide.

[0189] Certain compounds, including oligonucleotides In certain embodiments, the compounds described herein may be antisense compounds. In certain embodiments, the antisense compounds comprise or consist of oligomeric compounds. In certain embodiments, the oligomeric compounds comprise oligonucleotides, such as modified oligonucleotides. In certain embodiments, the modified oligonucleotides have a nucleobase sequence complementary to the nucleobase sequence of a target nucleic acid.

[0190] In certain embodiments, the compounds described herein comprise or consist of modified oligonucleotides, hi certain embodiments, the modified oligonucleotides have a nucleobase sequence that is complementary to the nucleobase sequence of a target nucleic acid.

[0191] In certain embodiments, the compound or antisense compound is single-stranded. Such single-stranded compounds or antisense compounds comprise or consist of oligomeric compounds. In certain embodiments, such oligomeric compounds comprise or consist of an oligonucleotide and optionally a conjugate group. In certain embodiments, the oligonucleotide is an antisense oligonucleotide. In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of the single-stranded antisense compound or oligomeric compound comprises a self-complementary nucleobase sequence.

[0192] In certain embodiments, the compound is double-stranded. Such double-stranded compounds include a first modified oligonucleotide having a region complementary to a target nucleic acid and a second modified oligonucleotide having a region complementary to the first modified oligonucleotide. In certain embodiments, the modified oligonucleotide is an RNA oligonucleotide. In such embodiments, thymine nucleobases in the modified oligonucleotide are replaced with uracil nucleobases. 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 first modified oligonucleotide is 12 to 30 linked nucleosides in length and the second modified oligonucleotide is 12 to 30 linked nucleosides in length. In certain embodiments, the antisense compound is double-stranded. Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid, and a second oligomeric compound having a region complementary to the first oligomeric compound.The first oligomeric compound of such double-stranded antisense compounds generally comprises or consists 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.Either or both oligomeric compounds of the double-stranded antisense compounds can comprise a conjugate group.The oligomeric compound of the double-stranded antisense compounds can comprise non-complementary overhanging nucleosides.

[0193] In certain embodiments, the compound comprises a 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 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 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.

[0194] Examples of single-stranded and double-stranded compounds include, but are not limited to, oligonucleotides, siRNAs, microRNA targeting oligonucleotides, and single-stranded RNAi compounds such as small hairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNA mimics.

[0195] 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 target nucleic acid to which it is targeted.

[0196] In certain embodiments, the compounds described herein include oligonucleotides between 10 and 30 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 12 and 30 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 12 and 22 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 14 and 30 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 14 and 20 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 15 and 30 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 15 and 20 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 16 and 30 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 16 and 20 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 17 and 30 linked subunits in length. In certain embodiments, the compounds described herein include oligonucleotides between 17 and 20 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides between 18 and 30 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides between 18 and 21 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides between 18 and 20 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides between 20 and 30 linked subunits in length.In other words, such oligonucleotides are 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 in length, respectively. In certain embodiments, the compounds described herein comprise oligonucleotides 14 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides 16 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides 17 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides 18 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides 19 linked subunits in length. In certain embodiments, the compounds described herein comprise oligonucleotides 20 linked subunits in length. In another embodiment, the compounds described herein comprise oligonucleotides 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 oligonucleotides of linked subunit lengths 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, or a range defined by any two of the foregoing values.In some embodiments, the linking subunit is a nucleotide, a nucleoside, or a nucleobase.

[0197] In certain embodiments, the compound may further comprise additional features or elements, such as a conjugate group, attached to the oligonucleotide. In certain embodiments, such compounds are antisense compounds. In certain embodiments, such compounds are oligomeric compounds. In embodiments where the conjugate group comprises a nucleoside (i.e., the nucleoside that joins the conjugate group to the oligonucleotide), the nucleoside of the conjugate group is not counted in the length of the oligonucleotide.

[0198] In certain embodiments, the compound may be shortened or truncated. For example, a single subunit may 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 may delete two subunits from the 5' end of the compound, or alternatively, two subunits from the 3' end. Alternatively, the deleted nucleosides may be dispersed throughout the compound.

[0199] When a single additional subunit is present in the extended compound, the additional subunit can be located at the 5' or 3' end of the compound. When two or more additional subunits are present, the added subunits can be adjacent to each other, for example, two subunits can be added to the 5' end of the compound (5' addition) or alternatively to the 3' end (3' addition) of the compound. Alternatively, the added subunits can be dispersed throughout the compound.

[0200] It is possible to increase or decrease the length of a compound, such as an oligonucleotide, and / or introduce mismatch bases without eliminating activity (Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992; Gautschi et al. J. Natl. Cancer Inst. 93:463-471, March 2001; Maher and Dolnick Nuc. Acid. Res. 16:3341-3358, 1988). However, seemingly small changes in oligonucleotide sequence, chemical structure, and motif can result in large differences in one or more of the many properties required for clinical development (Seth et al. J. Med. Chem. 2009, 52, 10; Egli et al. J. Am. Chem. Soc. 2011, 133, 16642).

[0201] 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 function at least in part through the RISC pathway to degrade and / or sequester target nucleic acids (and thus include microRNA / microRNA mimic compounds). As used herein, the term siRNA is intended to be equivalent to other terms used to describe nucleic acid molecules that can mediate 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, and post-transcriptional gene silencing RNA (ptgsRNA). Furthermore, as used herein, the term "RNAi" is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, or epigenetics.

[0202] In certain embodiments, the first strand of the compound is an siRNA guide strand, and the second strand of the compound is an siRNA passenger strand. In certain embodiments, the second strand of the compound is complementary to the first strand. In certain embodiments, each strand of the compound is 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides in length. In certain embodiments, the first or second strand of the compound can comprise a conjugate group.

[0203] In certain embodiments, the compounds described herein include modified oligonucleotides. Certain modified oligonucleotides have one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric structures, which can be specified in terms of absolute stereochemistry as (R) or (S), such as α or β for sugar anomers or (D) or (L) for amino acids. The modified oligonucleotides provided herein include all such possible isomers, including their racemic and optically pure forms, unless otherwise specified. Similarly, all cis- and trans-isomers and tautomeric forms are also included.

[0204] The compounds described herein include variations in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated elements. For example, compounds herein containing hydrogen atoms may be 1 Isotopic substitutions encompassed by the compounds herein include, but are not limited to, all possible deuterium substitutions for H hydrogen atoms. 1 Instead of H 2 H or 3 H, 12 Instead of C 13 C or 14 C. 14 Instead of N 15 N, 16 Instead of O 17 O or 18 O and 32 Instead of S 33 S, 34 S, 35 S or 36In certain embodiments, non-radioactive isotope substitution can confer beneficial new properties to the compound for use as a therapeutic or research tool. In certain embodiments, radioactive isotope substitution can make the compound suitable for research or diagnostic purposes, such as imaging assays.

[0205] Specific Mechanism In certain embodiments, the compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds comprise oligomeric compounds. In certain embodiments, the compounds described herein can hybridize to target nucleic acids to produce at least one antisense activity. In certain embodiments, the compounds described herein selectively affect one or more target nucleic acids. Such compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acids to produce one or more desired antisense activities, and does not hybridize to one or more non-target nucleic acids, or does not hybridize to one or more non-target nucleic acids in a manner that results in undesirable significant antisense activity.

[0206] In certain antisense activities, hybridization of the compounds described herein to a target nucleic acid results in the recruitment of a protein that cleaves the target nucleic acid. For example, certain compounds described herein result in RNase H-mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex does not need to be unmodified DNA. In certain embodiments, the compounds described herein are sufficiently "DNA-like" to induce RNase H activity. Furthermore, in certain embodiments, one or more non-DNA-like nucleosides in the gap of a gapmer are tolerated.

[0207] In certain antisense activity, the compounds or some of the compounds described herein are loaded into RNA-induced silencing complex (RISC), which ultimately leads to the cleavage of target nucleic acid.For example, some of the compounds described herein lead to the cleavage of target nucleic acid by Argonaute.The compound loaded into RISC is an RNAi compound.RNAi compound can be double-stranded (siRNA) or single-stranded (ssRNA).

[0208] In certain embodiments, hybridization of a compound described herein to a target nucleic acid does not result in the recruitment of a protein that cleaves the target nucleic acid. In certain such embodiments, hybridization of a compound to a target nucleic acid results in perturbation of splicing of the target nucleic acid. In certain such embodiments, hybridization of a compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of a compound to a target nucleic acid results in perturbation of translation of the target nucleic acid.

[0209] Antisense activity can be observed directly or indirectly, hi certain embodiments, observing or detecting antisense activity comprises 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 splice variants of a nucleic acid or protein, and / or a phenotypic change in a cell or animal.

[0210] Target nucleic acids, target regions and nucleotide sequences In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide comprising a region complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid 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, which comprise introns, exons, and untranslated regions. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is located entirely within an intron. In certain embodiments, the target region spans an intron / exon junction. In certain embodiments, the target region is located at least 50% within an intron. In certain embodiments, the target nucleic acid is located in a cell expressing a GLP-1 receptor. In certain embodiments, the GLP-1 receptor-expressing cell is a pancreatic cell, such as a pancreatic beta islet cell.

[0211] Hybridization In some embodiments, hybridization occurs between a compound disclosed herein and a target nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of nucleic acid molecules.

[0212] Hybridization can occur under varying conditions, which are sequence-dependent and determined by the nature and composition of the nucleic acid molecules to be hybridized.

[0213] Methods for determining whether a sequence specifically hybridizes to a target nucleic acid are well known in the art. In certain embodiments, the compounds provided herein are capable of specifically hybridizing to a target nucleic acid.

[0214] Complementarity An oligonucleotide is said to be complementary to another nucleic acid if the nucleobase sequence of such oligonucleotide, or one or more regions thereof, matches the nucleobase sequence of another oligonucleotide or nucleic acid, or one or more regions thereof, when the two nucleobase sequences are aligned in reverse. Nucleobase matches or complementary nucleobases described herein are limited to the following 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 specified. Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at each nucleoside and may contain one or more nucleobase mismatches. An oligonucleotide is fully complementary, or 100%, if such an oligonucleotide has a nucleobase match at each nucleoside without any nucleobase mismatches.

[0215] In certain embodiments, the compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds comprise oligomeric compounds. Non-complementary nucleobases between the compound and the target nucleic acid can be tolerated as long as they remain capable of specifically hybridizing to the target nucleic acid. Furthermore, the compound can hybridize across one or more segments of the target nucleic acid, so that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structures, mismatches, or hairpin structures).

[0216] In certain embodiments, the compounds provided herein, or specified portions thereof, are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a target nucleic acid, target region, target segment, or specified portion thereof. In certain embodiments, the compounds provided herein, or specified portions thereof, are 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, or any range therein, complementary to a target nucleic acid, target region, target segment, or specified portion thereof. The percent complementarity of a compound with a target nucleic acid can be determined using routine methods.

[0217] For example, a compound in which 18 of the 20 nucleobases of the compound are complementary to the target region and therefore specifically hybridize would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be clustered or interspersed between complementary nucleobases and do not need to be contiguous to each other or to complementary nucleobases. Thus, an 18-nucleobase compound having four non-complementary nucleobases flanking two regions that are completely complementary to the target nucleic acid would have an overall complementarity of 77.8% with the target nucleic acid. The percent complementarity of a compound with a region of a target nucleic acid can be routinely determined using BLAST (Basic Local Alignment Search Tool) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) known in the art. Percent homology, percent sequence identity, or percent complementarity can be determined, for example, using the default settings of the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0218] In certain embodiments, the compounds described herein or specific portions thereof are fully complementary (i.e., 100% complementary) to a target nucleic acid or a specific portion thereof. For example, a compound can be fully complementary to a target nucleic acid or a target region, segment, or sequence thereof. As used herein, "fully complementary" means that each nucleobase of the compound is complementary to a corresponding nucleobase of the target nucleic acid. For example, a 20-nucleobase compound is fully complementary to a target sequence that is 400 nucleobases long, provided there is a 20-nucleobase portion of the target nucleic acid that is fully complementary to the compound. "Fully complementary" can also be used in reference to a specific portion of a first and / or second nucleic acid. For example, a 20-nucleobase portion of a 30-nucleobase compound can be "fully complementary" to a target sequence that is 400 nucleobases long. A 20-nucleobase portion of a 30-nucleobase compound is fully complementary to a target sequence if the target sequence has a corresponding 20-nucleobase portion in which each nucleobase is complementary to the 20-nucleobase portion of the compound. At the same time, the entire 30 nucleobase compound may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleobases of the compound are complementary to the target sequence.

[0219] In certain embodiments, the compounds described herein contain one or more nucleobases mismatched to a target nucleic acid. In certain such embodiments, such mismatches reduce antisense activity against the target, but reduce activity against non-targets even more. Thus, in certain such embodiments, the selectivity of the compound is improved. In certain such embodiments, the mismatches are specifically located within an oligonucleotide having a gapmer motif. In certain such embodiments, the mismatches are located at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5' end of the gap region. In certain such embodiments, the mismatches are located at positions 9, 8, 7, 6, 5, 4, 3, 2, or 1 from the 3' end of the gap region. In certain such embodiments, the mismatches are located at positions 1, 2, 3, or 4 from the 5' end of the wing region. In certain such embodiments, the mismatches are located at positions 4, 3, 2, or 1 from the 3' end of the wing region. In certain such embodiments, the mismatches are located specifically within an oligonucleotide not having a gapmer 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' end 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' end of the oligonucleotide.

[0220] The location of the non-complementary nucleobase can be at the 5'-end or 3'-end of the compound. Alternatively, one or more non-complementary nucleobases can be at an internal position of the compound. When two or more non-complementary nucleobases are present, they can be contiguous (i.e., linked) or non-contiguous. In one embodiment, the non-complementary nucleobase is located in the wing segment of a gapmer oligonucleotide.

[0221] In certain embodiments, compounds described herein that are 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length, or that are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length, comprise no more than four, no more than three, no more than two, or no more than one non-complementary nucleobase to a target nucleic acid, e.g., a target nucleic acid or a defined portion thereof.

[0222] In certain embodiments, compounds described herein that are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length, or that are 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, comprise 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 to a target nucleic acid, e.g., a target nucleic acid or a defined portion thereof.

[0223] In certain embodiments, the compounds described herein also include those that are complementary to a portion of a target nucleic acid. As used herein, a "portion" refers to a defined number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases of a compound. In certain embodiments, a compound is complementary to a portion of at least 8 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 9 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 10 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 11 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 12 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 13 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 14 nucleobases of a target segment. In certain embodiments, a compound is complementary to a portion of at least 15 nucleobases of a target segment. In certain embodiments, the compound is complementary to a portion of at least 16 nucleobases of a target segment. Compounds complementary to a portion of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobases of a target segment, or a range defined by any two of these values, are also contemplated.

[0224] identity The compounds provided herein may also have a specified percent identity to a compound or portion thereof represented by a specific nucleotide sequence, SEQ ID NO:, or specific ISIS or ION number. In certain embodiments, the compounds described herein are antisense compounds or oligomeric compounds. In certain embodiments, the compounds described herein are modified oligonucleotides. A compound used herein is identical to a sequence disclosed herein if it has the same nucleic acid base pairing ability. For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shorter and longer versions of the compounds described herein, as well as compounds with non-identical bases to the compounds provided herein, are also contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the compound. The percent identity of a compound is calculated according to the number of bases that share identical base pairs with the sequence to which it is compared.

[0225] In certain embodiments, the compounds described herein or portions thereof are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of the compounds or SEQ ID NOs or portions thereof disclosed herein. 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 a specific nucleotide sequence, SEQ ID NO, or a compound represented by a specific ISIS or ION number, or portion thereof, or any percentage therebetween, and the compounds include oligonucleotides 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' end 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' end of the oligonucleotide.

[0226] In certain embodiments, the compounds described herein comprise or consist of antisense compounds. In certain embodiments, the portion of the antisense compound is compared to the same length portion of the target nucleic acid. In certain embodiments, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleobase portion is compared to the same length portion of the target nucleic acid.

[0227] In certain embodiments, the compounds described herein comprise or consist of oligonucleotides. In certain embodiments, a portion of the oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

[0228] Specific modified compounds In certain embodiments, the compounds described herein comprise or consist of oligonucleotides composed of linked nucleosides. The oligonucleotides can be unmodified oligonucleotides (RNA or DNA) or modified oligonucleotides. Modified oligonucleotides contain at least one modification relative to unmodified RNA or DNA (i.e., at least one modified nucleoside (including a modified sugar moiety and / or a modified nucleobase) and / or at least one modified internucleoside linkage).

[0229] A. Modified Nucleosides A modified nucleoside comprises a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

[0230] 1. Modified sugar moiety 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 surrogates may contain one or more substitutions that correspond to substitutions in other types of modified sugar moieties.

[0231] In certain embodiments, the modified sugar moiety comprises a non-bicyclic modified sugar moiety comprising a furanosyl ring bearing one or more acyclic substituents, including, but not limited to, substituents at the 2', 4', and / or 5' positions. In certain embodiments, one or more acyclic substituents of the non-bicyclic modified sugar moiety are branched. Examples of suitable 2'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2'-substituent is halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C 10 Alkoxy, O-C1~C 10 Substituted alkoxy, O-C1-C 10 Alkyl, O-C1-C 10 Substituted alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ) or OCH2C(=O)-N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10Alkyl and 2'-substituents described in U.S. Patent Nos. 6,531,584 to Cook et al.; 5,859,221 to Cook et al.; and 6,005,087 to Cook et al. Certain embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro(NO), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of suitable 4'-substituents for linear non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in WO 2015 / 106128 to Manoharan et al. Examples of suitable 5'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, non-bicyclic modified sugars include two or more non-bridging sugar substituents, such as 2'-F-5'-methyl sugar moieties and modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008 / 101157 and Rajeev et al., U.S. Patent Application Publication No. 2013 / 0203836.

[0232] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside is 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 )) wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 It is alkyl.

[0233] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety that includes a linear 2'-substituent selected from F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N-(CH3)2, and OCH2C(=O)-N(H)CH3 ("NMA").

[0234] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety that includes a linear 2'-substituent selected from F, OCH3, and OCH2CH2OCH3.

[0235] Nucleosides containing modified sugar moieties, e.g., non-bicyclic modified sugar moieties, are referred to by the position of the substitution on the sugar moiety of the nucleoside. For example, nucleosides containing 2'-substituted or 2-modified sugar moieties are referred to as 2'-substituted nucleosides or 2-modified nucleosides.

[0236] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring to result in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to, 4'-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 the S configuration, referred to as "hindered ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CHOCH3)-O-2' ("hindered MOE" or "cMOE") and analogs thereof (e.g., U.S. Pat. No. 7,399,845 to Seth et al., U.S. Pat. No. 7,569,686 to Bhat et al., U.S. Pat. No. 7,569,686 to Swayze et al., U.S. Pat. No. 7,399,845 to Seth et al., U.S. Pat. No. 7,569,686 to Swayze ... No. 7,741,457 to Seth et al. and U.S. Pat. No. 8,022,193 to Swayze et al.), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., U.S. Pat. No. 8,278,283 to Seth et al.), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., U.S. Pat. No. 8,278,425 to Prakash et al.), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Pat. No. 7,696,345 to Allerson et al. and U.S. Pat. No. 8,124,745 to Allerson et al.), 4'-CH2-C(H)(CH3)-2' (see, e.g., U.S. Pat. No. 8,124,745 to Zhou, et al. al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see, e.g., U.S. Pat. No. 8,278,426 to Seth et al.), 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 C1-C 12alkyl) (see, for example, U.S. Pat. No. 7,427,672 to Imanishi et al.).

[0237] In certain embodiments, such 4' to 2' bridges are independently -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x - and -N(R a )-, wherein: x is 0, 1 or 2; n is 1, 2, 3 or 4; Each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each of J1 and J2 is independently H, C1 to C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl or a protecting group.

[0238] Freier et al., Nucleic Acids, 2013-05-20 10:00:00 Research,1997,25(22),4429-4443、Albaek et al.,J.Org.Chem.,2006,71,7731-7740、Singh et al.,Chem.Commun.,1998,4,455-456;Koshkin et al. al.,Tetrahedron,1998,54,3607-3630;Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638;Kumar et al.,Bioorg.Med.Chem.Lett.,1998,8,219-2222; 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,2005,515;Braasch et al. al.,Chem.Biol.,2001,8,1-7;Orum et al.,Curr.Opinion Mol.Ther., 2001, 3,239-243; U.S. Patent No. 7,053,207 to Wengel et al., U.S. Patent No. 6,268,490 to Imanishi et al., U.S. Patent No. 6,770,748 to Imanishi et al., U.S. Patent Re. 44,779 to Imanishi et al.; U.S. Patent No. 6,794,499 to Wengel et al., U.S. Patent No. 6,670,461 to Wengel et al.; U.S. Patent No. No. 7,034,133 to Wengel et al., U.S. Pat. No. 8,080,644 to Wengel et al., U.S. Pat. No. 8,034,909 to Wengel et al., U.S. Pat. No. 8,153,365 to Wengel et al., U.S. Pat. No. 7,572,582 to Wengel et al., and U.S. Pat. No. 6,525,191 to Ramasamy et al., WO 2004 / 106356 to Torsten et al., WO 2004 / 106356 to Wengel et al. No. 91999 / 014226; WO 2007 / 134181 to Seth et al.; U.S. Pat. No. 7,547,684 to Seth et al.; U.S. Pat. No. 7,666,854 to Seth et al.; U.S. Pat. No. 8,088,746 to Seth et al.; U.S. Pat. No. 7,750,131 to Seth et al.; U.S. Pat. No. 8,030,467 to Seth et al.; U.S. Pat. No. 8,268,980 to Seth et al. No. 8,546,556 to Seth et al.; U.S. Patent No. 8,530,640 to Seth et al.; U.S. Patent No. 9,012,421 to Migawa et al.; U.S. Patent No. 8,501,805 to Seth et al.; and U.S. Patent Application Publication Nos. 2008 / 0039618 to Allerson et al. and 2015 / 0191727 to Migawa et al.

[0239] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by their isomeric configuration. For example, LNA nucleosides (described herein) can be in the α-L or β-D configuration. [ka] α-L-methyleneoxy (4'-CH2-O-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that have demonstrated antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this specification, a general description of a bicyclic nucleoside includes both isomeric configurations. In exemplary embodiments herein, when the position of a particular bicyclic nucleoside (e.g., LNA or cEt) is specified, it is in the β-D configuration unless otherwise specified.

[0240] In certain embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (eg, 5'-substituted and 4'-2' bridging sugars).

[0241] In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain such embodiments, an oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described herein. For example, certain sugar surrogates include substitutions at the 4'-sulfur atom and the 2'-position (see, e.g., U.S. Pat. Nos. 7,875,733 to Bhat et al. and 7,939,677 to Bhat et al.) and / or the 5'-position.

[0242] In certain embodiments, the sugar surrogate comprises a ring with more than five atoms. For example, in certain embodiments, the sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans can be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), mannitol nucleic acid ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA: [ka] ("F-HNA", see, e.g., Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. Nos. 8,440,803; and Swayze et al., U.S. Pat. No. 9,005,906. F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran), and of the formula: [ka] wherein, independently for each of said modified THP nucleosides: Bx is a nucleobase moiety; T3 and T4 are each independently an internucleoside linking group that joins the modified THP nucleoside to the remainder of the oligonucleotide, or one of T3 and T4 is an internucleoside linking group that joins the modified THP nucleoside to the remainder of the oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked 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, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Each of R1 and R2 is independently selected from hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where X is O, S, or NJ1, and each J1, J2, and J3 is independently H or C1-C6 alkyl. and nucleosides containing additional modified THP compounds having the formula:

[0243] In certain embodiments, modified THP nucleosides are provided wherein q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, 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.

[0244] In certain embodiments, the sugar surrogate comprises a ring having six or more atoms and two or more heteroatoms. For example, its use in nucleosides and oligonucleotides containing morpholino sugar moieties has been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Patent No. 5,698,685; Summerton et al., U.S. Patent No. 5,166,315; Summerton et al., U.S. Patent No. 5,185,444; and Summerton et al., U.S. Patent No. 5,034,506). As used herein, the term "morpholino" refers to the following structure: [ka] In certain embodiments, morpholinos can be modified, for example, by adding or varying substituents from the morpholino structure above. Such sugar surrogates are referred to herein as "modified morpholinos."

[0245] In certain embodiments, the sugar surrogate comprises an acyclic moiety. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include, but are not limited to, peptide nucleic acids ("PNAs"), acyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and the nucleosides and oligonucleotides described in Manoharan et al., WO 2011 / 133876.

[0246] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

[0247] Modified nucleobases The modification or substitution of nucleobase (or base) is structurally distinct from naturally occurring or synthetic unmodified nucleobase, but functionally interchangeable with naturally occurring or synthetic unmodified nucleobase.Natural nucleobase and modified nucleobase can both participate in hydrogen bonding.Such nucleobase modification can confer nuclease stability, binding affinity or some other beneficial biological properties to antisense compounds.

[0248] In certain embodiments, the compound described herein comprises modified oligonucleotide.In certain embodiments, modified oligonucleotide comprises one or more nucleosides that comprise unmodified nucleobase.In certain embodiments, modified oligonucleotide comprises one or more nucleosides that comprise modified nucleobase.In certain embodiments, modified oligonucleotide comprises one or more nucleosides that do not comprise nucleobase, which are referred to as abasic nucleosides.

[0249] In certain embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines, alkyl- or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6 substituted purines. In certain embodiments, 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-thiolalkyl, 8-hydroxymethyl ... cytosine, 8-aza and other 8-substituted purines, 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp).Modified nucleobases can also include 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 U.S. Pat. No. 3,687,808 to Merigan et al., The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; and Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

[0250] Publications that teach the preparation of certain of the above and other modified nucleobases include, but are not limited to, Manoharan et al., U.S. Patent Application Publication No. 2003 / 0158403; Manoharan et al., U.S. Patent Application Publication No. 2003 / 0175906; Dinh et al., U.S. Patent No. 4,845,205; Spielvogel et al., U.S. Patent No. 5,130,302; Rogers et al., U.S. Patent No. 5,134,066; Bischofberger et al., U.S. Patent No. 5,134,066; No. 175,273 to Urdea et al.; U.S. Pat. No. 5,367,066 to Urdea et al.; U.S. Pat. No. 5,432,272 to Benner et al.; U.S. Pat. No. 5,434,257 to Matteucci et al.; U.S. Pat. No. 5,457,187 to Gmeiner et al.; U.S. Pat. No. 5,459,255 to Cook et al.; U.S. Pat. No. 5,484,908 to Froehler et al.; U.S. Pat. No. 5,502,177 to Matteucci et al.; U.S. Pat. No. 5,525,711 to Hawkins et al. ;Haralambidis et al., U.S. Patent No. 5,552,540;Cook et al., U.S. Patent No. 5,587,469;Froehler et al., U.S. Patent No. 5,594,121;Switzer et al., U.S. Patent No. 5,596,091;Cook et al., U.S. Patent No. 5,614,617;Froehler et al., U.S. Patent No. 5,645,985;Cook et al., U.S. Patent No. 5,681,941;Cook et al., U.S. Patent No. 5,811,534;Cook et al., U.S. Patent No. 5, No. 750,692 to Cook et al.; U.S. Pat. No. 5,948,903 to Cook et al.; U.S. Pat. No. 5,587,470 to Cook et al.; U.S. Pat. No. 5,457,191 to Cook et al.; U.S. Pat. No. 5,763,588 to Matteucci et al.; U.S. Pat. No. 5,830,653 to Froehler et al.; U.S. Pat. No. 5,808,027 to Cook et al.; U.S. Pat. No. 6,166,199 to Cook et al.; and U.S. Pat. No. 6,005,096 to Matteucci et al.

[0251] In certain embodiments, the compound that targets 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.

[0252] 3. Modified internucleotide linkages The naturally occurring internucleoside linkage in RNA and DNA is a 3' to 5' phosphodiester linkage. In certain embodiments, compounds described herein having one or more modified (i.e., non-naturally occurring) internucleoside linkages are often selected over compounds having naturally occurring internucleoside linkages due to desirable properties such as, for example, improved cellular uptake, improved affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0253] In certain embodiments, the compound that targets the target nucleic acid comprises one or more modified internucleoside linkages.In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages.In certain embodiments, each internucleoside linkage of antisense compounds is phosphorothioate internucleoside linkage.

[0254] In certain embodiments, the compounds described herein include oligonucleotides. Oligonucleotides with modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.

[0255] In certain embodiments, the nucleosides of modified oligonucleotides can be linked to each other using any internucleoside linkage. Two main classes 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), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S"), and phosphorodithioates ("HS-P=S"), containing phosphodiester linkages ("P=O"). Representative non-phosphorus-containing internucleoside linkage groups include, but are not limited to, methylenemethylimino (-CH-N(CH)-O-CH-), thiodiester, thionocarbamate (-OC(=O)(NH)-S-); siloxane (-O-SiH-O-); and N,N'-dimethylhydrazine (-CH-N(CH)-N(CH)-). Modified internucleoside linkages can be used to vary, and generally increase, the nuclease resistance of oligonucleotides compared to naturally occurring phosphate linkages. In certain embodiments, internucleoside linkages containing chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0256] 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 including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, e.g., "Carbohydrate Modifications in Antisense Research"; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH2 moieties.

[0257] In certain embodiments, an oligonucleotide comprises modified internucleoside linkages arranged along the oligonucleotide or a region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, the internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of the two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments, the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. Because the nucleoside motifs are independently selected, such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif, and if a gapped nucleoside motif is present, the wing length and gap length may or may not be the same.

[0258] In certain embodiments, the oligonucleotide comprises a region having an alternating internucleoside linkage motif. In certain embodiments, the oligonucleotide comprises a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate, and at least one internucleoside linkage is phosphorothioate.

[0259] In certain embodiments, an oligonucleotide comprises at least six phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least eight phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least ten phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least six consecutive phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least eight consecutive phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least ten consecutive phosphorothioate internucleoside linkages. 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 three nucleotides of the 3' end of the oligonucleotide.

[0260] In certain embodiments, the oligonucleotide comprises one or more methyl phosponate linkages. In certain embodiments, the oligonucleotide having a gapmer nucleoside motif comprises a linkage motif that comprises all phosphorothioate linkages except for one or two methyl phosponate linkages. In certain embodiments, one methyl phosponate is present in the central gap of the oligonucleotide having a gapmer nucleoside motif.

[0261] In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages can be reduced and the number of phosphodiester internucleoside linkages can be increased. In certain embodiments, the number of phosphorothioate internucleoside linkages can be reduced and the number of phosphodiester internucleoside linkages can be increased while still maintaining nuclease resistance. In certain embodiments, it is desirable to reduce the number of phosphorothioate internucleoside linkages while maintaining nuclease resistance. In certain embodiments, it is desirable to increase the number of phosphodiester internucleoside linkages while maintaining nuclease activity.

[0262] 4. Specific motifs In certain embodiments, the compounds described herein comprise oligonucleotides. Oligonucleotides can have motifs, such as patterns of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkages. In such embodiments, the modified, unmodified, and differentially modified sugar moieties, nucleobases, and / or internucleoside linkages of the modified oligonucleotides define a pattern or motif. In certain embodiments, the sugar moieties, nucleobases, and internucleoside linkage patterns are each independent of one another. Thus, modified oligonucleotides can be described by their sugar motif, nucleobase motif, and / or internucleoside linkage motif (as used herein, nucleobase motif describes nucleobase modification independent of the nucleobase sequence).

[0263] 1. Specific glycomotifs In certain embodiments, the compounds described herein comprise oligonucleotides. In certain embodiments, the oligonucleotides comprise one or more types of modified sugar and / or unmodified sugar moieties arranged along the oligonucleotide or regions thereof in defined patterns or sugar motifs. In certain instances, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0264] In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two outer regions or "wings" and a central or internal region or "gap." The three regions of the gapmer motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides, with at least a portion of the sugar moiety of each nucleoside of the wing being different from at least a portion of the sugar moiety of the nucleoside of the gap. Specifically, at least the sugar moiety of the nucleoside of each wing closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) is different from the sugar moiety of the adjacent gap nucleoside, thus defining the boundary between the wing and the gap (i.e., the wing / gap junction). In certain embodiments, the sugar moieties within the gap are identical to each other. In certain embodiments, the gap contains one or more nucleosides having a sugar moiety that is different from the sugar moieties of one or more other nucleosides in the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric gapmers). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmers).

[0265] In certain embodiments, the gapmer wing comprises 1 to 5 nucleosides. In certain embodiments, the gapmer wing comprises 2 to 5 nucleosides. In certain embodiments, the gapmer wing comprises 3 to 5 nucleosides. In certain embodiments, all of the nucleosides in the gapmer are modified nucleosides.

[0266] In certain embodiments, the gapmer gap contains 7 to 12 nucleosides. In certain embodiments, the gapmer gap contains 7 to 10 nucleosides. In certain embodiments, the gapmer gap contains 8 to 10 nucleosides. In certain embodiments, the gapmer gap contains 10 nucleosides. In certain embodiments, each nucleoside in the gapmer gap is an unmodified 2'-deoxynucleoside.

[0267] In certain embodiments, the gapmer is a deoxygapmer. In such embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2'-deoxynucleosides and the nucleosides on the wing side of each wing / gap junction are modified nucleosides. In certain such embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In certain such embodiments, each nucleoside of each wing is a modified nucleoside.

[0268] In certain embodiments, a modified oligonucleotide has a fully modified sugar motif, and each nucleoside of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, a modified oligonucleotide comprises or consists of a region having a fully modified sugar motif, and each nucleoside of the region contains a modified sugar moiety. In certain embodiments, a modified oligonucleotide comprises or consists of a region having a fully modified sugar motif, and each nucleoside within the fully modified region contains the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of the uniform modification contains the same 2'-modification.

[0269] 2. Specific nucleobase motifs In certain embodiments, the compounds described herein comprise oligonucleotides. In certain embodiments, the oligonucleotides comprise modified and / or unmodified nucleobases arranged in a defined pattern or motif along the oligonucleotide or a region thereof. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methylcytosine.

[0270] In certain embodiments, a 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 of the 3'-end of the oligonucleotide. In certain embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 5'-end of the oligonucleotide.

[0271] In certain embodiments, an oligonucleotide having a gapmer motif comprises a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is present in the central gap of the oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2'-deoxyribosyl moiety. In certain embodiments, the modified nucleobase is selected from 2-thiopyrimidine and 5-propynepyrimidine.

[0272] 3. Specific internucleoside linkage motifs In certain embodiments, the compounds described herein comprise oligonucleotides. In certain embodiments, the oligonucleotides comprise modified and / or unmodified internucleoside linkages arranged along the oligonucleotide or regions thereof in a defined pattern or motif. In certain embodiments, essentially each internucleoside linkage group is a phosphate internucleoside linkage (P=O). In certain embodiments, each internucleoside linkage group of a modified oligonucleotide is phosphorothioate (P=S). In certain embodiments, each internucleoside linkage group of a modified oligonucleotide is independently selected from phosphorothioate and phosphate internucleoside linkages. In certain embodiments, the sugar motif of the modified oligonucleotide is a gapmer, and all internucleoside linkages within the gap are modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphate linkages. In certain embodiments, the terminal internucleoside linkage is modified.

[0273] 5. Specific Modified Oligonucleotides In certain embodiments, the compounds described herein comprise modified oligonucleotides. In certain embodiments, the above-described modifications (sugar, nucleobase, internucleoside linkage) are incorporated into the modified oligonucleotide. In certain embodiments, the modified oligonucleotide is characterized by its modification, motif, and total length. In certain embodiments, such parameters are independent of each other. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif can be modified or unmodified, and may or may not follow the gapmer modification pattern of sugar modification. For example, the internucleoside linkages within the wing regions of a sugar gapmer can be identical or different from each other and may be identical or different from the internucleoside linkages in the gap region of the sugar motif. Similarly, such gapmer oligonucleotides can contain one or more modified nucleobases independent of the gapmer pattern of sugar modification. Furthermore, in certain instances, oligonucleotides are described by a total length or range and by the length or length range of two or more regions (e.g., regions of nucleosides with specific sugar modifications). In such cases, it may be possible to select the number of each range that results in an oligonucleotide having a total length that does not fall within the specified range. In such cases, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions A, B, and C, where region A consists of 2-6 linked nucleosides having a particular sugar motif, region B consists of 6-10 linked nucleosides having a particular sugar motif, and region C consists of 2-6 linked nucleosides having a particular sugar motif. Such embodiments do not include modified oligonucleotides in which A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (although these numbers of nucleosides are possible within the requirements for A, B, and C). This is because the total length of such an oligonucleotide would be 22, exceeding the upper limit (20) for the total length of a modified oligonucleotide.In this specification, if a description of an oligonucleotide is not provided with respect to one or more parameters, those parameters are not limited. Thus, a modified oligonucleotide that is described solely as having a gapmer sugar motif without further description can have any length, internucleoside linkage motif, and nucleobase motif. Unless otherwise indicated, all modifications are independent of the nucleobase sequence.

[0274] Certain conjugated compounds In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide (modified or unmodified) and, optionally, one or more conjugate groups and / or terminal groups. A conjugate group consists of one or more conjugate moieties and a conjugate linker that connects the conjugate moieties to the oligonucleotide. A conjugate group can be attached to either or both termini of the oligonucleotide and / or at any internal position. In certain embodiments, a conjugate group is attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, a conjugate group attached to either or both termini of the oligonucleotide is a terminal group. In certain such embodiments, a conjugate group or terminal group is attached to the 3' and / or 5' termini of the oligonucleotide. In certain such embodiments, a conjugate group (or terminal group) is attached to the 3' terminus of the oligonucleotide. In certain embodiments, a conjugate group is attached near the 3' terminus of the oligonucleotide. In certain embodiments, a conjugate group (or terminal group) is attached to the 5' terminus of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 5' end of the oligonucleotide.

[0275] Examples of terminal groups include, but are not limited to, a conjugate group, a capping group, a phosphate moiety, a protecting group, a modified or unmodified nucleoside, and two or more nucleosides, independently modified or unmodified.

[0276] GLP-1 receptor ligand conjugate moiety In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 receptor ligand conjugate moiety. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 receptor ligand conjugate moiety. In certain embodiments, the conjugate linker connects the GLP-1 receptor ligand conjugate moiety to the oligonucleotide. In certain embodiments, the oligonucleotide is a modified oligonucleotide. In certain embodiments, the GLP-1 receptor ligand conjugate moiety comprises a small molecule, an aptamer, an antibody, or a peptide.

[0277] 1. Specific GLP-1 receptor small molecule conjugate moiety In certain embodiments, the compound comprises an oligonucleotide and a small molecule conjugate moiety capable of binding to the GLP-1 receptor. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a small molecule conjugate moiety capable of binding to the GLP-1 receptor. In certain embodiments, the oligonucleotide is a modified oligonucleotide.

[0278] Any small molecule conjugate moiety known in the art that is capable of binding to the GLP-1 receptor can be used in some embodiments. For example, in certain embodiments, the small molecule conjugate moiety capable of binding to the GLP-1 receptor may be any of the small molecule conjugate moieties 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 peptide 1 receptor," PNAS 2007 Jan 16;104(3):937-42; or Wang et al., "Non-peptidic glucose-like peptide-1 receptor agonists: aftermath of a serendipitous discovery," Acta Pharmacologica Sinica (2010) 31:1026-1030, which are incorporated herein by reference in their entirety.

[0279] In certain embodiments, the small molecule conjugate moiety capable of binding to the GLP-1 receptor has the following formula: [ka] [ka] [ka] The device has one of the following:

[0280] 2. Specific GLP-1 receptor antibody conjugate moiety In certain embodiments, the compound comprises an oligonucleotide and an antibody or fragment thereof capable of binding to the GLP-1 receptor. 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. In certain embodiments, the oligonucleotide is a modified oligonucleotide. Any antibody or fragment thereof known in the art capable of binding to the GLP-1 receptor can be used in some embodiments. In certain embodiments, the compound comprises an oligonucleotide and an antibody or fragment thereof capable of binding to the GLP-1 receptor described in WO2005018536, U.S. Patent Application Publication No. 20060275288, U.S. Patent No. 8,389,689, or WO2011056644 (which are incorporated herein by reference in their entireties). 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 as described in WO2005018536, U.S. Patent Application Publication No. 20060275288, U.S. Patent No. 8,389,689, or WO2011056644, which are incorporated herein by reference in their entireties.

[0281] 3. Specific GLP-1 peptide conjugate moieties In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide or a fragment or variant thereof. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide or a fragment or variant thereof. In certain embodiments, the oligonucleotide is a modified oligonucleotide. Any GLP-1 peptide or a fragment or variant thereof known in the art can be used in some embodiments. In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide described in U.S. Patent Application Publication No. 20140206607; U.S. Patent No. 9,187,522; U.S. Patent No. 8,329,419; or WO 2007 / 124461 (which are incorporated herein by reference in their entireties). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide described in U.S. Patent Application Publication No. 20140206607; U.S. Patent No. 9,187,522; U.S. Patent No. 8,329,419; or WO 2007 / 124461, which are incorporated by reference in their entireties.

[0282] In certain embodiments, the compound comprises an oligonucleotide and GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which in the conventional three letter notation 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 (sequence and a GLP-1 peptide conjugate moiety 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 or 31 consecutive amino acids that 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 an equal length portion of the amino acid sequence of In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety 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, or 31 consecutive amino acids 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 an equal-length portion of the amino acid sequence of GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (SEQ ID NO: 1), where NH2 represents the C-terminal amide.

[0283] In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which, in the conventional three letter notation, 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- and a GLP-1 peptide conjugate moiety 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 or 31 consecutive amino acids 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 an equal length portion of the amino acid sequence of Gly (SEQ ID NO: 1). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety 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, or 31 consecutive amino acids 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 an equal-length portion of the amino acid sequence of GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (SEQ ID NO: 1), where NH2 represents the C-terminal amide.

[0284] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety 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, or 31 consecutive amino acids 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 an equal 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 moiety 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, or 31 consecutive amino acids 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 an equal-length portion of the amino acid sequence of GLP-1(7-37).

[0285] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety of 8 to 50 amino acids in length that 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 over its entire length 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 moiety of 8 to 50 amino acids in length that 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 over its entire length to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0286] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety of 8 to 50 amino acids in length that 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 over its entire length 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 moiety of 8 to 50 amino acids in length that 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 over its entire length to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0287] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety comprising 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 moiety comprising the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0288] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety consisting 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 moiety consisting of the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1).

[0289] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety comprising the amino acid sequence of GLP-1(7-36)amide: HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR-NH2 (which in the conventional three letter code 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 moiety comprising the amino acid sequence of GLP-1(7-36)amide:HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR-NH2 (which in the conventional three letter notation 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 moiety comprising the amino acid sequence of GLP-1(7-36):HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR, which in the conventional three letter code 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 moiety comprising the amino acid sequence of GLP-1(7-36):HAEGTFTSDV SSYLEGQAAKEFIAWLVKGR, which in the conventional three letter code 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).

[0290] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety 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 moiety 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 moiety 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 moiety consisting of the amino acid sequence of GLP-1(7-36) (SEQ ID NO:2).

[0291] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety comprising the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety comprising the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), where NH2 represents the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety comprising the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety comprising the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), where NH2 represents the C-terminal amide.

[0292] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety consisting of the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety consisting of the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), where NH2 represents the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety consisting of the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety consisting of the amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKG-NH2 (SEQ ID NO: 3), where NH2 represents the C-terminal amide.

[0293] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety comprising the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety comprising the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), where NH2 represents the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety comprising the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety comprising the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), where NH2 represents the C-terminal amide.

[0294] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety consisting of the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety consisting of the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), where NH2 represents the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety consisting of the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG (which in conventional three letter notation 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 moiety consisting of the amino acid sequence: EGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 4), where NH2 represents the C-terminal amide.

[0295] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a compound selected from the group consisting of, but not limited to, liraglutide (VICTOZA® from Novo Nordisk); albiglutide (SYNCRIA® from GlaxoSmithKline); taspoglutide (Hoffman La-Roche); LY2189265 (Eli Lilly and Company); LY2428757 (Eli Lilly and Company); 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(7-37); -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: 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.

[0296] 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 iraglutide, taspoglutide, exenatide, lixisenatide, and 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 diabetes," Bioorg Med Chem Lett. 2013 Jul 15;23(14):4011-8, which is incorporated herein by reference in its entirety.

[0297] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: H-AibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO: 22), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the 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.

[0298] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: H-AibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSX-NH2 (SEQ ID NO: 23), where Aib is aminoisobutyric acid, X is penicillamine, and NH2 represents the C-terminal amide. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the 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.

[0299] In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker, and the amino acid sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC (which in the conventional three letter notation 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 a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker, and the amino acid sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRC-NH2 (SEQ ID NO: 24), where NH2 indicates the C-terminal amide.

[0300] In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker, and the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 25). In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker, and the 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), where H represents the N-terminus and NH2 represents the C-terminal amide.

[0301] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: AGEGTFTSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPSC, which in the conventional three letter notation 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-Ser-Cys (SEQ ID NO: 26). In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: AGEGTFTSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO: 26), where NH2 represents the C-terminal amide.

[0302] In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker and the amino acid sequence: AGEGTFTSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSX, where X is penicillamine (which in the conventional three letter notation 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-Ser-Pen (SEQ ID NO: 27)), where Pen is penacillamine. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: AGEGTFTSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPPSX-NH2 (SEQ ID NO: 27), where X is penicillamine and NH2 represents the C-terminal amide.

[0303] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLV (which in the conventional three letter notation 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)), where Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLV-NH2 (SEQ ID NO: 28), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0304] In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker, and the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVK (which in the conventional three letter notation 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)), where Aib is aminoisobutyric acid. In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety comprising or consisting of an oligonucleotide, optionally a conjugate linker, and the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVK-NH2 (SEQ ID NO: 29), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0305] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKG (which in the conventional three letter notation 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)), where Aib is aminoisobutyric acid. In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKG-NH2 (SEQ ID NO: 30), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0306] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGG (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGG-NH2 (SEQ ID NO: 31), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0307] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGP (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGP-NH2, (SEQ ID NO: 32), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0308] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPS (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPS-NH2 (SEQ ID NO: 33), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0309] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSS (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSS-NH2 (SEQ ID NO: 34), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0310] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSG (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSG-NH2 (SEQ ID NO: 35), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0311] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGA (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGA-NH2 (SEQ ID NO: 36), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0312] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAP (which in the conventional three letter notation 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 moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAP-NH2 (SEQ ID NO: 37), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0313] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0314] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSZ-NH2 (SEQ ID NO: 38), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0315] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRGZ, which in the conventional three letter notation 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 [ka] It is 4-azidonorleucine containing

[0316] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRGZ-NH2 (SEQ ID NO: 39), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0317] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEGQAANXEFIAWLVRGRG, which in the conventional three letter notation 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 a group of the formula: [ka] It is lysine (5-azidopentanoic acid amide) having the formula:

[0318] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEGQAANXEFIAWLVRGRG-NH2 (SEQ ID NO: 40), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and X or Xaa is a group of the formula: [ka] It is lysine (5-azidopentanoic acid amide) having the formula:

[0319] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVK-AibRZ, which in the conventional three letter notation 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 [ka] It is 4-azidonorleucine containing

[0320] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEGQAAKEFIAWLVK-AibRZ-NH2 (SEQ ID NO: 41), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0321] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0322] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRZ-NH2 (SEQ ID NO: 42), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0323] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPZ, which in the conventional three letter notation 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 [ka] It is 4-azidonorleucine containing

[0324] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPZ-NH2 (SEQ ID NO: 43), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0325] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSZ, which in the conventional three letter notation 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 [ka] It is 4-azidonorleucine containing

[0326] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSZ-NH2 (SEQ ID NO: 44), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0327] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKZ, which in the conventional three letter notation 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 [ka] It is 4-azidonorleucine containing

[0328] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKZ-NH2 (SEQ ID NO: 45), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0329] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVZ, which in the conventional three letter notation 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 [ka] It is 4-azidonorleucine containing

[0330] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVZ-NH2 (SEQ ID NO: 46), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0331] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVC (which in the conventional three letter code 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.

[0332] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVC-NH2 (SEQ ID NO: 47), where Aib is aminoisobutyric acid and NH2 represents the C-terminal amide.

[0333] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLZ, which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0334] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLZ-NH2 (SEQ ID NO: 48), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0335] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWZ, which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0336] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWZ-NH2 (SEQ ID NO: 49), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0337] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAZ, which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0338] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAZ-NH2 (SEQ ID NO: 50), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0339] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0340] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGZ-NH2 (SEQ ID NO: 51), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0341] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0342] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNZ-NH2 (SEQ ID NO: 52), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0343] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0344] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKZ-NH2 (SEQ ID NO: 53), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0345] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0346] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLZ-NH2 (SEQ ID NO: 54), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0347] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWZ (which in the conventional three letter code 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 [ka] It is 4-azidonorleucine containing

[0348] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWZ-NH2 (SEQ ID NO: 55), where NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0349] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSZ, which in the conventional three letter notation 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-Ser-Zaa (SEQ ID NO: 56), wherein Aib is aminoisobutyric acid and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0350] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSZ-NH2 (SEQ ID NO: 56), where Aib is aminoisobutyric acid, NH2 represents the C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine containing

[0351] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC, which in the conventional three letter notation 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).

[0352] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC-NH2 (SEQ ID NO: 57), where NH2 represents the C-terminal amide.

[0353] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety 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, or 31 consecutive amino acids 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 an equal-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 moiety 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, or 31 consecutive amino acids 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 an equal-length portion of the amino acid sequence of any one of SEQ ID NOs: 1-57.

[0354] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety of 8 to 50 amino acids in length that 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 over its entire length to the amino acid sequence of any one of SEQ ID NOs: 1 to 57. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety of 8 to 50 amino acids in length that 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 over its entire length to the amino acid sequence of any one of SEQ ID NOs: 1 to 57.

[0355] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety of 8 to 50 amino acids in length that 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 over its entire length to the amino acid sequence of any one of SEQ ID NOs: 1 to 57. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety of 8 to 50 amino acids in length that 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 over its entire length to the amino acid sequence of any one of SEQ ID NOs: 1 to 57.

[0356] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety 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 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 moiety 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 GLP-1(7-37) (SEQ ID NO: 1).

[0357] In certain embodiments, a compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 1-57. In certain embodiments, a compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety 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 of SEQ ID NOs: 1-57. In certain embodiments, a compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety 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 of SEQ ID NOs: 1-57.

[0358] In any of the above embodiments, the GLP-1 peptide conjugate moiety may comprise a conservative amino acid substitution, amino acid analog, or amino acid derivative. In certain embodiments, conservative amino acid substitutions include replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of serine with threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing an amide group; exchange of a basic residue with another basic residue; or replacement of an aromatic residue with another aromatic residue, or combinations 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.

[0359] Additional GLP-1 peptide conjugate moieties or analogs that can be used in the embodiments provided herein are described in U.S. Patent Application Publication No. 20140206607; U.S. Patent No. 9,187,522; WO 2007 / 124461; WO 2014 / 096179; WO 2009 / 030738; WO 2016 / 055610; and U.S. Patent No. 8,329,419, all of which are incorporated herein by reference in their entireties.

[0360] Conjugate Linker In certain embodiments, a 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 the conjugate linker by a single bond. In certain embodiments, the conjugate linker comprises a chain structure such as a hydrocarbyl chain or an oligomer of repeating units such as ethylene glycol, nucleoside, or amino acid units.

[0361] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amido, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amido, and ether groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amido groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0362] In certain embodiments, conjugate linkers, such as those described above, are known in the art to be useful for attaching a conjugate group to a bifunctional linking moiety, such as a parent compound, such as an oligonucleotide provided herein. Generally, a bifunctional linking moiety contains at least two functional groups. One functional group is selected to bind to a specific site on the compound, and the other functional group is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, a bifunctional linking moiety contains one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0363] Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-merimidomethyl)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, a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0364] In certain embodiments, the conjugate linker comprises 1 to 10 linker-nucleosides. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments, such linker-nucleosides comprise modified sugar moieties. In certain embodiments, the linker-nucleosides are unmodified. In certain embodiments, the linker-nucleosides optionally comprise a protected heterocyclic base selected from a purine, a substituted purine, a pyrimidine, or a substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. Generally, it is desirable for the linker-nucleoside to be cleaved from the compound after reaching the target tissue. Thus, the linker-nucleosides are generally attached to each other and to the remainder of the compound via a cleavable bond. In certain embodiments, such a cleavable bond is a phosphodiester bond.

[0365] As used herein, linker-nucleosides are not considered part of the oligonucleotide. Thus, in embodiments where a compound comprises an oligonucleotide consisting of a specific number or range of linked nucleosides and / or a specific percent complementarity to a reference nucleic acid, and the compound also comprises a conjugate group containing a conjugate linker comprising a linker-nucleoside, the linker-nucleoside is not counted toward the length of the oligonucleotide and is not used to determine the percent complementarity of the oligonucleotide to the reference nucleic acid. For example, a compound may comprise (1) a modified oligonucleotide consisting of 8 to 30 nucleosides and (2) a conjugate group comprising 1 to 10 linker-nucleosides contiguous with a nucleoside of the modified oligonucleotide. The total number of contiguous linked nucleosides in such a compound is greater than 30. Alternatively, a compound may comprise a modified oligonucleotide consisting of 8 to 30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such a compound is 30 or less. Unless otherwise indicated, a conjugate linker contains 10 or fewer linker-nucleosides. In certain embodiments, a conjugate linker contains 5 or fewer linker-nucleosides. In certain embodiments, a conjugate linker contains 3 or fewer linker-nucleosides. In certain embodiments, a conjugate linker contains 2 or fewer linker-nucleosides. In certain embodiments, a conjugate linker contains 1 or fewer linker-nucleoside.

[0366] In certain embodiments, it is desirable for the conjugate group to be cleaved from the oligonucleotide. For example, in certain situations, compounds containing certain conjugate moieties are better absorbed by certain cell types, and it is desirable for the conjugate group to be cleaved to release the unconjugated or parent oligonucleotide once the compound is absorbed. Therefore, certain conjugates may generally contain one or more cleavable moieties within the conjugate linker. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety is a group of atoms containing at least one cleavable bond. In certain embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four, or five or more cleavable bonds. In certain embodiments, the cleavable moiety is selectively cleaved within a cell or an intracellular compartment such as a lysosome. In certain embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme such as a nuclease.

[0367] In certain embodiments, the cleavable bond is selected from among an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, the cleavable bond is one or both esters of a phosphodiester. In certain embodiments, the cleavable moiety comprises a phosphate or a phosphodiester. In certain embodiments, the cleavable moiety is a phosphate bond between the oligonucleotide and the conjugate moiety or conjugate group.

[0368] In certain embodiments, the cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, the one or more linker-nucleosides are bonded to each other and / or to the remainder of the compound via a cleavable bond. In certain embodiments, such cleavable bond is an unmodified phosphodiester bond. In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside attached to either the 3'- or 5'-terminal nucleoside of the oligonucleotide by a phosphate internucleoside bond and covalently bonded to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate bond. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.

[0369] 1. Specific hexylamino linker In certain embodiments, the compound has the following structure: [ka] wherein each n is independently selected from 0, 1, 2, 3, 4, 5, 6, or 7. The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0370] In certain embodiments, the compound has the following structure: [ka] wherein each n is independently 1 to 20; and p is 1 to 6. The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0371] In certain embodiments, the compound has the following structure: [ka] (wherein each n is independently 1 to 20) The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0372] In certain embodiments, the compound has the following structure: [ka] (wherein each n is independently 1 to 20) The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0373] In certain embodiments, the compound has the following structure: [ka] The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0374] In certain embodiments, the compound has the following structure: [ka] The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0375] In certain embodiments, the compound has the following structure: [ka] The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker selected from:

[0376] In certain embodiments, the compound has the following structure: [ka] The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker having the formula:

[0377] In certain embodiments, the compound has the following structure: [ka] (In the ceremony X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety; and Y is directly or indirectly linked to the modified oligonucleotide. The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker having the formula:

[0378] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by any of the conjugate linkers described in WO 2014 / 179620, which is incorporated herein by reference in its entirety.

[0379] 2. Specific alkyl phosphate linkers In certain embodiments, the compound has the following structure: [ka] (In the formula: the phosphate group is linked to the modified oligonucleotide and Y is linked to the conjugate group; Y is a phosphodiester or amino (—NH—) group; Z is a group of the formula: [ka] is a pyrrolidinyl group having the formula: j is 0 or 1; n is from about 1 to about 10; p is 1 to about 10; m is 0 or 1 to 4; and When Y is amino, m is 1. The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker having the formula:

[0380] In certain embodiments, Y is amino (—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 2 to 10 and p is 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 is a group of the formula: [ka] It has.

[0381] In certain embodiments, n is 2 and p is 3. In certain embodiments, n is 5 and p is 6.

[0382] In certain embodiments, the compound has the following structure: [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety; and T1 comprises a modified oligonucleotide; and Bx is a modified or unmodified nucleobase. The oligonucleotide comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker having the formula:

[0383] 3. Specific Click Chemistry Linkers In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, and the conjugate linker is prepared using click chemistry known in the art. The compound is prepared using click chemistry, in which the alkynyl phosphonate internucleoside linkage on the oligomeric compound attached to a solid support is converted to a 1,2,3-triazolyl phosphonate internucleoside linkage, 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 use in some embodiments can be prepared by click chemistry as described in "Click Chemistry for Biotechnology and Materials Science," Ed. Joerg Laham, Wiley 2009 (which is incorporated herein by reference in its entirety).

[0384] In certain embodiments, a click reaction is used to [ka] with, but not limited to, the following compounds: [ka] wherein Y is attached directly or indirectly to the oligonucleotide. with an oligonucleotide having a terminal amine, comprising [ka] which is reacted with an azide-bearing GLP-1 receptor ligand conjugate moiety to give: [ka] where N—N=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety. By obtaining the above, the GLP-1 receptor ligand conjugate moiety can be linked to the oligonucleotide.

[0385] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker being one of the following compounds: [ka] It is prepared from

[0386] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker being [ka] Includes.

[0387] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker being [ka] Includes.

[0388] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] wherein N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0389] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] wherein N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0390] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] wherein N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0391] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, wherein the conjugate linker is prepared using click chemistry and a disulfide bond.

[0392] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] where N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly 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 Includes.

[0393] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] where N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly 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 Includes.

[0394] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] where N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly 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 Includes.

[0395] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] where N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the remainder of the GLP-1 receptor ligand conjugate moiety; m is 1; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0396] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the compound comprising: [ka] where N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly 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 Includes.

[0397] 4. Certain Maleimide and Maleimide Acid Linkers In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker being [ka] Including, X is directly or indirectly attached to the GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to the oligonucleotide.

[0398] In certain embodiments, the conjugate linker described above is capable of linking a peptide to an oligonucleotide. In certain embodiments, the compound comprises an oligonucleotide linked to a peptide by a conjugate linker, the conjugate linker being: [ka] Including, X is attached directly or indirectly to the peptide; and Y is attached directly or indirectly to the oligonucleotide.

[0399] In certain embodiments, the compound comprises an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] (In the formula, R=(CH2) n and n is 1 to 12; X is attached directly or indirectly to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0400] In certain embodiments, the compound comprises an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] (In the formula, [ka] and; m is 1 to 12; X is attached directly or indirectly to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0401] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, the first compound comprising an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] (In the formula, R=(CH2) n and n is 1 to 12; X is attached directly or indirectly to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker comprising [ka] (In the formula, R=(CH2) n and n is 1 to 12; X is attached directly or indirectly to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0402] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, the first compound comprising an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] (In the formula, [ka] and; m is 1 to 12; X is attached directly or indirectly to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker comprising [ka] (In the formula, [ka] and m is 1 to 12; X is attached directly or indirectly to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0403] In certain embodiments, the compound comprises an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0404] In certain embodiments, the compound comprises an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0405] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, the first compound comprising an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker being [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide and the second compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety, such as a peptide, by a conjugate linker, the conjugate linker comprising [ka] wherein X is directly or indirectly attached to a GLP-1 receptor ligand conjugate moiety, such as a peptide; and Y is attached directly or indirectly to the oligonucleotide Includes.

[0406] 5. Specific disulfide bonds In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, wherein the conjugate linker comprises a disulfide bond. In certain embodiments, the oligonucleotide comprises an activated disulfide that forms a disulfide bond with the GLP-1 peptide conjugate moiety. In certain embodiments, the compound comprises an oligonucleotide comprising an activated disulfide moiety capable of forming a cleavable or reversible bond with the GLP-1 peptide conjugate moiety. In certain embodiments, the compound comprises an oligonucleotide directly attached to the GLP-1 peptide conjugate moiety by a disulfide bond without a conjugate linker.

[0407] In certain embodiments, the compound includes a linker between the oligonucleotide and the activated disulfide moiety. In another embodiment, the activated 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 activated disulfide moieties are methanethiosulfonate and dithiocarbomethoxy. In a further embodiment, the activated disulfide is substituted or unsubstituted dithiopyridyl, substituted or unsubstituted dithiobenzothiazolyl, or substituted or unsubstituted dithiotetrazolyl. Preferred activated 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-dithio-1-methyl-1H-tetrazolyl.

[0408] In some embodiments, the activated disulfide moiety has the formula -SS(O) n -R1, wherein n is 0, 1 or 2; and R1 is selected from substituted or unsubstituted heterocyclic, substituted or unsubstituted aliphatic, or -C(O)O-R2, where R2- is substituted or unsubstituted aliphatic.

[0409] In another embodiment, the activated disulfide moiety has the formula -SS(O)2-substituted or unsubstituted C1-C 12 Alkyl or -SSC(O)O- substituted or unsubstituted C1-C 12 In certain embodiments, the activated disulfide moiety includes methanethiosulfonate and dithiocarbomethoxy. In further embodiments, the activated disulfide can be substituted or unsubstituted dithiopyridyl, substituted or unsubstituted dithiobenzothiazolyl, or substituted or unsubstituted dithiotetrazolyl. Further examples of activated 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.

[0410] In some embodiments, the divalent linking group is a divalent substituted or unsubstituted aliphatic group. In other embodiments, the divalent linking group has the formula -Q1-G-Q2-, where: Q1 and Q2 are independently absent or substituted or unsubstituted C1 to C 12 Alkylene, substituted or unsubstituted alkarylene or -(CH2) m -O-(CH2) p - selected from each m and p is independently an integer from 1 to about 10; 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-.

[0411] Examples of divalent linking groups include, but are not limited to: [ka] Examples include:

[0412] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 peptide conjugate moiety by a disulfide bond as described in U.S. Patent No. 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 U.S. Patent No. 7,713,944, which is incorporated herein by reference in its entirety.

[0413] In certain embodiments, any of the above-described compounds comprising an oligonucleotide linked to a GLP-1 peptide conjugate moiety by a disulfide bond, either directly or via a conjugate linker described herein, can comprise a disulfide bond between the cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β-cyclopentamethylenepropionic acid moiety of the GLP-1 peptide conjugate moiety and the oligonucleotide or the conjugate linker. In certain embodiments, the compound comprises an oligonucleotide linked directly to the GLP-1 peptide conjugate moiety by a disulfide bond. In certain embodiments, the compound comprises an oligonucleotide linked directly to the GLP-1 peptide conjugate moiety 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 moiety of the GLP-1 peptide conjugate moiety. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety, wherein a disulfide bond connects the conjugate linker and the GLP-1 peptide conjugate moiety, and wherein 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 moiety, wherein a disulfide bond connects the conjugate linker to a cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid moiety of the GLP-1 peptide conjugate moiety, and wherein the oligonucleotide is attached to the conjugate linker. In certain embodiments, the cysteine, penicillamine, homocysteine, mercaptopropionic acid or β-mercapto-β,β,-cyclopentamethylenepropionic acid moiety is present at the N-terminus, C-terminus, side chain or internal amino acid position of the GLP-1 peptide conjugate moiety.

[0414] 6. Specific enzyme-cleavable bonds In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker comprising 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.

[0415] Enzymes known in the art for use in activating prodrugs can be used to cleave the enzyme-cleavable moiety 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-glutamyltransferase, β-glucuronidase, β-lactamase, alkaline phosphatase, aminopeptidase, penicillin amidase, or nitroreductase.

[0416] In certain embodiments, the enzyme-cleavable moiety is cleavable by a protease or peptidase. In certain embodiments, the enzyme-cleavable moiety is selected from the group consisting of gastricsin, memapsin-2, chymosin, renin, renin-2, cathepsin D, cathepsin E, penicillopepsin, rhizopuspepsin, mucorpepsin, variepsin, aspergillopepsin I, endothiapepsin, saccharopepsin, phytepsin, plasmepsin-1, plasmepsin-2, yapsin-1, yapsin-2, nepenthesin, memapsin-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 1 retropepsin, Mason-Pfizer leukemia virus retropepsin, human endogenous retropepsin, and the like. Virus K retropepsin, retropepsin (human T-cell leukemia virus), bovine leukemia virus retropepsin, Rous sarcoma virus retropepsin, scytalido glutamic peptidase, aspergilloglutamic peptidase, thermopsin, signal peptidase II, spumapepsin, type 4 prepilin peptidase 1, omptin, plasminogen activator Pla, papain, chymopapain, caricain, glycyl endopeptidase, stem bromelain, ficain, actinidain, cathepsin V, vignain, cathepsin X, zingipain, cathepsin F, ananain, fruit bromelain, cathepsin L, cathepsin L1 (Fasciola hepatica) sp.), cathepsin S, cathepsin K, cathepsin H, aleurene, histrisain, cathepsin B, dipeptidyl-peptidase I, peptidase 1 (mite), CPB peptidase, cruzipain, V-cath peptidase, bleomycin hydrolase (animal), bleomycin hydrolase (yeast), aminopeptidase C, CPC peptidase, calpain-1, calpain-2, calpain-3, Tpr peptidase (Porphyromonas gingivalis), poliovirus type picornain 3C,Hepatitis A virus type picornain 3C, human rhinovirus type 2 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, adenine, potato virus type Y helper component peptidase, Sindbis virus type nsP2 peptidase, streptopain, clostripain, ubiquitinyl hydrolase-L1, ubiquitinyl hydrolase-L3, legumain ( Plant β-type), legumain, animal type, caspase-1, caspase-3, caspase-7, caspase-6, caspase-8, caspase-9, pyroglutamyl-peptidase I (prokaryotes), pyroglutamyl-peptidase I (chordates), mouse hepatitis coronavirus papain-like peptidase 1, ubiquitin-specific peptidase 5, tymovirus peptidase, rabbit hemorrhagic disease virus 3C-like peptidase, gingipain RgpA, gingipain Kgp, γ-glutamyl hydrolase, foot-and-mouth disease virus L-peptidase, porcine infectious gastroenteritis virus Illus-type main peptidase, calicivirin, staphopain A, Ulp1 peptidase, separase (yeast type), YopJ protein, PfpI peptidase, sortase A (Staphylococcus type), aminopeptidase N, lysyl aminopeptidase (bacteria), aminopeptidase A, leukotriene A4 hydrolase, pyroglutamyl-peptidase II, cytosolic alanyl aminopeptidase, cystinyl aminopeptidase, aminopeptidase B, aminopeptidase Ey, angiotensin-converting enzyme peptidase unit 1, peptidyl-dipeptidase Acer, angiotensin-converting enzyme peptidase unit 2, angiotensin-converting enzyme-2, thimeto-oligopeptidase, neurolysin, saccharolysin, oligopeptidase A, peptidyl-dipeptidase Dcp, mitochondrial intermediate peptidase, oligopeptidase F, thermolysin, vibriolysin, pseudolysin, coccolysin, aureolysin, stearolysin, mycolysin, snaparicin,Reishimanolysin, bacterial collagenase V, bacterial collagenase G / A, matrix metallopeptidase-1, matrix metallopeptidase-8, matrix metallopeptidase-2, matrix metallopeptidase-9, matrix metallopeptidase-3, matrix metallopeptidase-10 (Human sapiens type), matrix metallopeptidase-11, matrix metallopeptidase-7, matrix metallopeptidase-12, envelysin, matrix metallopeptidase-13, membrane-type matrix metallopeptidase-1, membrane-type matrix metallopeptidase-2, matrix metallopeptidase-20, fragilisin, matrix metallopeptidase-26, serralysin, aeruginolysin, gametolysin, astacin, meprin α-subunit, procollagen C-peptidase, coriolisin L, coriolisin H, fluvastasin, fibrolase, jalaragin, adamalysin, atrolysin A, atrolysin B, atrolysin C, atrolysin 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, neprilysin, endothelin-converting enzyme 1, oligopeptidase O1, neprilysin-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, pitorilysin, insulysin, mitochondrial processing peptidase β-subunit, nardilysin, leucine aminopeptidase 3, leucyl aminopeptidase (plant type), aminopeptidase I, aspartyl aminopeptidase, membrane dipeptidase, glutamate carboxypeptidase, peptidase T, carboxypeptidase Ss1, β-lytic metallopeptidase, staphylolysin, lysostaphin, methionyl aminopeptidase 1 (Escherichia type), methionyl aminopeptidase 2, Xaa-Pro dipeptidase (bacterial type), aminopeptidase P (bacterial), aminopeptidase P2, Xaa-Pro dipeptidase IgA1-specific metallopeptidase, tentoxylysin, vonoxylysin, aminopeptidase Y, aminopeptidase Ap1, aminopeptidase S (Streptomyces type), glutamate carboxypeptidase II, carboxypeptidase Taq, anthrax lethal factor, deuterolysin, peptidyl-Lys methyltransferase Talopeptidase, FtsH peptidase, m-AAA peptidase, i-AAA peptidase, AtFtsH2 peptidase, paparisin-1, Ste24 peptidase, dipeptidyl-peptidase III, site 2 peptidase, sporulation factor SpoIVFB, HybD peptidase, gpr peptidase, chymotrypsin A (cattle type), granzyme B (human sapiens type), factor VII-activated peptidase, trypsin (Streptomyces griseus type), hypodermin C, elastase-2, cathepsin G, myeloblastin, granzyme A, granzyme M, chymase (human (Homo sapiens) type), mast cell peptidase 1 (Rattus type), duodenase, tryptase α, granzyme K, mast cell peptidase 5 (mouse numbering), trypsin 1, chymotrypsin B, elastase-1, pancreatic endopeptidase E, pancreatic elastase II, enteropeptidase, chymotrypsin C, prostasin,Kallikrein 1, kallikrein-related peptidase 2, kallikrein-related peptidase 3, kallikrein 1 (house mouse (Mus musculus)), kallikrein 1-related peptidase b3, kallikrein 1-related peptidase c2 (Rattus norvegicus), kallikrein 13 (house mouse (Mus musculus)), ancrod, thrombin, complement factor D, activated complement component C1r, activated complement component C1s, complement factor Bb, mannan-binding lectin-associated serine peptidase 1, complement factor I, coagulation factor XIIa, plasma kallikrein, coagulation factor XIa, coagulation factor IXa, coagulation factor VIIa, coagulation factor Xa, thrombin, protein C (activated), coagulation factor C (horseshoe crab (Limulus), horseshoe crab (Tachypleus)), activated, coagulation factor B ( Limulus, Tachypleus), activation, clotting enzyme (Tachypleus type), acrosin, hepsin, mannan-binding lectin-associated serine peptidase 2, urokinase-type plasminogen activator, t-plasminogen activator, plasmin, kallikrein-related peptidase 6, plasminogen activator (Desmodus type), kallikrein-related peptidase 8, Kallikrein-related peptidase 4, streptoglycin A, streptoglycin B, streptoglycin E, α-lytic endopeptidase, glutamyl peptidase I, DegP peptidase, HtrA2 peptidase, lysyl endopeptidase (bacterial), kallikrein-related peptidase 7, matriptase, togavirin, IgA1-specific serine peptidase (Neisseria type), flavinvirin, subtilisin Carlsberg, subtilisin lentus (lentu s), thermolysin, subtilisin Ak1, lactosepin I, C5a peptidase, dentilisin, subtilisin BPN', subtilisin E, aqualysin 1, cerevisiae, origin, 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), acylaminoacyl-peptidase, fibroblast activation protein α subunit, oligopeptidase B, carboxypeptidase Y, serine carboxypeptidase A, serine carboxypeptidase C, serine carboxypeptidase, peptidase D, 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, assemblins, cytomegalovirus assemblins, herpesvirus type 8 assemblins, repressor LexA, UmuD protein , signal peptidase I, mitochondrial inner membrane peptidase 1, signal peptidase SipS, signalase (animal) 21 kDa component, lysosomal Pro-Xaa carboxypeptidase, dipeptidyl-peptidase II, hepacivillin, potyvirus P1 peptidase, pestivirus NS3 polyprotein peptidase, equine arteritis virus serine peptidase, prolyl aminopeptidase, C-terminal processing peptidase-1, C-terminal processing peptidase-2, trichorne core peptidase (archaea), signal peptide Peptidase A, infectious pancreatic necrosis birnavirus Vp4 peptidase, dipeptidase E, sedolisin, sedolisin-B, tripeptidyl-peptidase I, kumamolisin, physalolisin, SpoIVB peptidase, archaean proteasome, β component, bacterial proteasome, β component, HslV component of HslUV peptidase, constitutive proteasome catalytic subunit 1, constitutive proteasome catalytic subunit 2, constitutive proteasome catalytic subunit 3, γ-glutamyltransferase 1 (bacterial type), Murein tetrapeptidase LD-carboxypeptidase (Escherichia type), PepA aminopeptidase, presenilin 1, polypropepsin, canditropsin, candidapepsin SAP2, caspase-2, caspase DRONC (Drosophila melanogaster) type peptidase, ubiquitin-specific peptidase 7, human coronavirus 229E main peptidase, SARS coronavirus picornain 3C-like peptidase, AvrPphB peptidase, sortase B,The enzyme-cleavable moiety is cleavable by a protease or peptidase selected from psychrophilic alkaline metallopeptidase (Pseudomonas sp.), acutolysin A, aminopeptidase S (Staphylococcus type), carboxypeptidase Pfu, isoaspartyl dipeptidase (metallotype), D-aminopeptidase DppA, and murein endopeptidase. In certain embodiments, the enzyme-cleavable moiety is cleavable by a cathepsin protease or peptidase.

[0417] Compositions and methods of formulating pharmaceutical compositions The compounds described herein can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The composition and method of formulating the pharmaceutical composition will 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.

[0418] Certain embodiments provide pharmaceutical compositions comprising one or more compounds or salts thereof. In certain embodiments, the pharmaceutical composition comprises a compound described herein and a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises a sterile saline solution and one or more compounds described herein. In certain embodiments, such a pharmaceutical composition consists of a sterile saline solution and one or more compounds. In certain embodiments, the sterile saline is pharmaceutical-grade saline. In certain embodiments, the pharmaceutical composition comprises one or more compounds described herein and sterile water. In certain embodiments, the pharmaceutical composition consists of a compound described herein and sterile water. In certain embodiments, the sterile water is pharmaceutical-grade water. In certain embodiments, the pharmaceutical composition comprises one or more compounds described herein and phosphate-buffered saline (PBS). In certain embodiments, the pharmaceutical composition consists of one or more compounds described herein and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical-grade PBS.

[0419] Pharmaceutical compositions containing the compounds described herein include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotides that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to an animal, including a human. Particular embodiments depict pharmaceutically acceptable salts of the compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0420] Non-Limiting Disclosure and Incorporation by Reference While the particular compounds, compositions and methods described herein are described with specificity according to particular embodiments, the following examples serve merely to illustrate, and are not intended to limit, the compounds described herein.

[0421] Each reference cited herein, including but not limited to scientific literature, patent application publications, GenBank accession numbers, etc., is incorporated by reference in its entirety.

[0422] Although the sequence listing accompanying this application identifies each sequence as either "RNA" or "DNA" where appropriate, in practice, these sequences may be modified with any combination of chemical modifications. Those of skill in the art will readily recognize that designations such as "RNA" or "DNA" describing modified oligonucleotides are arbitrary in certain cases. For example, an oligonucleotide containing a nucleotide containing a 2'-OH sugar moiety and a thymine base may be described as a DNA with a modified sugar (a 2'-OH instead of the single 2'-H of DNA) or an RNA with a modified base (thymine (methylated uracil) instead of the uracil of RNA). Thus, the nucleic acid sequences provided herein, including but not limited to those included in the sequence listing, are intended to encompass nucleic acids comprising any combination of natural or modified RNA and / or DNA, including, but not limited to, nucleic acids with such modified nucleobases. By way of further example, and without limitation, an oligomeric compound having the nucleobase sequence "ATCGATCG" encompasses any oligomeric compound having such a nucleobase sequence, whether modified or unmodified, including, but not limited to, compounds containing RNA bases, such as those having the sequence "AUCGAUCG," as well as compounds containing some DNA bases and some RNA bases, such as "AUCGATCG," and oligomeric compounds with other modified nucleobases, such as "ATmCGAUCG" (where mC represents a cytosine base containing a methyl group at the 5-position).

[0423] The compounds described herein include (R) or (S), such as α or β, as in the case of sugar anomers, or (D) or (L), as in the case of amino acids. The compounds provided herein include all such possible isomers, including their racemic and optically pure forms, unless otherwise specified. Also included are all cis- and trans-isomers and tautomeric forms. The compounds described herein include chirally pure or enriched mixtures and racemic mixtures. For example, oligonucleotides having multiple phosphorothioate internucleoside linkages include such compounds in which the chirality of the phosphorothioate internucleoside linkages is controlled or random.

[0424] Unless otherwise indicated, any compound, including oligomeric compounds, described herein includes pharmaceutically acceptable salts thereof.

[0425] The compounds described herein include variations in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated elements. For example, compounds herein containing hydrogen atoms may be 1 Isotopic substitutions encompassed by the compounds herein include, but are not limited to, all possible deuterium substitutions for H hydrogen atoms. 1 Instead of H 2 H or 3 H, 12 Instead of C 13 C or 14 C. 14 Instead of N 15 N, 16 Instead of O 17 O or 18 O and 32 Instead of S 33 S, 34 S, 35 S or 36 Examples include S. [Example]

[0426] Example 1: Preparation of antisense oligonucleotides (ASOs) targeting MALAT1 conjugated with GLP-1 peptides A method for preparing a conjugated modified oligonucleotide comprising GLP-1 conjugated at the 5' position via a 3-mercaptopropionate linker.

[0427] Unless otherwise stated, all reagents and solutions used in the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid supports are used for the incorporation of nucleoside residues, including, for example, T, A, G, and mC residues. 0.1 M solutions of phosphoramidites in anhydrous acetonitrile were used for 2'-deoxyribonucleosides, cEt BNA nucleosides, and appropriately protected 6-amino-hexanols. [ka] 5'-Hexylamino-modified oligonucleotide (ISIS 786434) (nucleobase sequence: TCAGCATTCTAATAGCAGC (SEQ ID NO: 38)) was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5'-end of the modified oligonucleotide contains a hexamethylene linker and a terminal amine. Compound 1 (3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) was obtained from Chem-Impex (cat# 11566). The modified oligonucleotide (approximately 6 μmol) was dissolved in 125 μL sodium phosphate buffer, 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. The reaction was complete after 2-3 hours, and product 2 was purified by HPLC on Source 30Q resin using Buffer A 100 mM NHOAc / 30% ACN / HO and Buffer B 100 mM NHOAc / 30% ACN / HO + 1.5M HCl. The product fractions were concentrated and stored at -20°C.

[0428] Compound 2 was used as the starting material for reaction with the GLP-1 peptide HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGlyProSerSerAlaProProProSerCys-NH2 (SEQ ID NO: 22), synthesized by standard solid-phase peptide synthesis. Aib is 2-aminoisobutyric acid. Compound 2 was dissolved in degassed water and the pH was adjusted to approximately 8.0 by adding 0.1 M NaHCO3. The GLP-1 peptide was dissolved in 50 / 50 0.1 M NaHCO3 (pH 8):DMF (dimethylformamide). The peptide solution was added to Compound 2 in small aliquots (30% of the total volume each time) at 5-minute intervals. After approximately 1 hour, 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 using Buffer A 100 mM NHOAc / 30% ACN / HO and Buffer B 100 mM NHOAc / 30% ACN / HO + 1.5 M NaBr. The product fractions were desalted by HPLC on a reverse-phase column to give ISIS 816385.

[0429] Example 2: Preparation of antisense oligonucleotides (ASOs) targeting MALAT1 conjugated with GLP-1 peptides A method for preparing a conjugated modified oligonucleotide comprising GLP-1 conjugated at the 5' position to a C-terminal penicillamine via a 3-mercaptopropionate linker. [ka] Compound 2 was synthesized as in Example 1 and used as the starting material for reaction with the GLP-1 peptide HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGlyProSerSerAlaProProProSerPen-NH2 (SEQ ID NO: 23), synthesized by standard solid-phase peptide synthesis. Aib is 2-aminoisobutyric acid, and Pen is penicillamine. Compound 2 was dissolved in degassed water, and the pH was adjusted to approximately 8.0 by adding 0.1 M NaHCO3. The GLP-1 peptide was dissolved in degassed water. The compound 2 solution and the peptide solution were mixed by gentle vortexing, and the pH was checked. The pH was adjusted to approximately 7.5 by adding 0.1 M NaHCO3. After approximately 2 hours, additional peptide was added, and the pH was adjusted upward by adding NaHCO3. The reaction was allowed to proceed for approximately 65 hours at 4° C. and the product was purified by HPLC as described in Example 1.

[0430] Example 3: Preparation of antisense oligonucleotides (ASOs) targeting FOXO1 conjugated with GLP-1 peptides A method for preparing a conjugated modified oligonucleotide comprising GLP-1 conjugated at the 5' position via a 3-mercaptopropionate linker.

[0431] ION 913193, a 5′-GLP-1 peptide-conjugated ASO targeting FOXO1, was prepared according to the procedure of Example 1, starting from a 5′-hexylamino-modified oligonucleotide (ION 913192) (nucleobase sequence: TCATCTTCTTAAAATACCC (SEQ ID NO: 59)) with the chemical modifications: Tdo mCdo Ado Tks mCks Tds Tds mCds Tds Tds Ads Ads Aks Tes Aks mCes mCks mCk (k=cEt; d=2′-deoxy; e=2′-MOE; mC=5-methylcytosine; o=phosphodiester; and s=phosphorothioate).

[0432] ION 913195, a control 5′-GLP-1 peptide-conjugated ASO with a nucleobase sequence mismatched to 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 the chemical modifications: Tdo mCdo Ado Gks Gks mCks mCds Ads 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).

[0433] Example 4: Preparation of insulin-targeted antisense oligonucleotides (ASOs) conjugated to GLP-1 peptides A method for preparing a conjugated modified oligonucleotide comprising GLP-1 conjugated at the 5' position via a mercaptopropionate linker.

[0434] ION 919553, an insulin-targeting 5′-GLP-1 peptide-conjugated ASO, was prepared according to the procedure of Example 1, starting from a 5′-hexylamino-modified oligonucleotide (ION 919553) (nucleobase sequence: TCAGCCAAGGTCTGAAGGTCACC (SEQ ID NO: 61)) with the chemical modifications: Tdo mCdo Ado Ges mCes mCes Aes Aes Gds Gds Tds mCds Tds Gds Ads Ads Gds Gds Tes mCes Aes mCes mCe (k=cEt; d=2′-deoxy; e=2′-MOE; mC=5-methylcytosine; o=phosphodiester; and s=phosphorothioate).

[0435] Example 5: Preparation of antisense oligonucleotides (ASOs) targeting MALAT1 conjugated with GLP-1 peptides ION 951976 (nucleobase sequence: GCTGCTATTAGAATGC (SEQ ID NO: 62)) with the chemical modifications: Ges mCeo Tdo Gdo mCdo Tdo Ado Tdo Tdo 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 with ION 951976 to generate a duplex of the two oligonucleotides.

[0436] Example 6: Specific targeting of pancreatic beta islet cells in vivo with GLP-1 peptide-conjugated ASO Research 1 To determine whether conjugation of a GLP-1 peptide to an ASO increases ASO delivery to the pancreas, chow-fed male C57BL / 6 mice received two intravenous injections of either the 3-10-3 cEt ASO (ISIS 556089) (nucleobase sequence: GCATTCTAATAGCAGC) (SEQ ID NO: 63) targeting murine MALAT1 or the 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.

[0437] MALAT1 expression was detected using the QuantiGene View RNA Tissue Assay (Affymetrix, cat. No. QVT0011). Species-specific MALAT1 probes were purchased from Affymetrix (cat. No. VB-11110-01 / mouse; VF1-13963 / monkey). Briefly, mouse tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4 mm sections. After deparaffinization, tissue slides were boiled in Affymetrix pretreatment solution for 10–30 min and then treated with protease at 40°C for 10–40 min, depending on the tissue. The MALAT1 RNA probe was used at a 1:40 dilution and incubated with the samples at 40°C for 120 min. After washing, the MALAT1 RNA / probe complex was hybridized with the preamplifier, amplifier, and AP-oligonucleotides at 40°C for 25, 15, and 15 minutes, respectively. After washing in PBS to remove free AP oligonucleotides, the slides were incubated with Fast Red substrate at room temperature for 30 minutes. Tissue images were obtained using an Aperio scanner. (Hung et al., 2013 Nuc Acid Ther. 369-78)

[0438] In situ hybridization analysis showed that GLP-1 peptide conjugation reduced MALAT1 staining in beta islet cells, but not in pancreatic acinar cells. ASO staining of pancreatic sections demonstrated that GLP-1 conjugation improved efficacy by increasing ASO delivery to the tissue. Mice treated with GLP-1-conjugated MALAT1 ASO (ISIS 816385) showed reduced MALAT1 expression in pancreatic beta islet cells, but mice treated with unconjugated MALAT1 ASO (ISIS 556089) did not. Mice treated with various doses of ISIS 816385 showed reduced MALAT1 expression in pancreatic beta islet cells. Mice treated with GLP-1-conjugated MALAT1 ASO (ISIS 816385) but not with unconjugated MALAT1 ASO (ISIS 556089) showed ASO accumulation in pancreatic beta islet cells. GLP-1-conjugated MALAT1 ASO (ISIS 816385) accumulated in pancreatic beta islet cells in a dose-dependent manner in treated mice.

[0439] Research 2 To determine the dose response of the GLP-1-conjugated MALAT1 ASO (ISIS 816385) described in Example 1 on pancreatic MALAT1 expression, chow-fed male C57BL / 6 mice received a single intravenous injection of ISIS 816385 or the unconjugated MALAT1 ASO (ISIS 556089) described above at concentrations of 0.2, 0.06, and 0.02 μmol / kg.

[0440] MALAT1 expression was detected using the QuantiGene View RNA tissue assay described above.

[0441] In situ hybridization analysis showed that GLP-1 peptide conjugation reduced MALAT1 staining in pancreatic beta islet cells at doses of 0.2 μmol / kg and 0.06 μmol / kg. No observable effects of ISIS 816385 or ISIS 556089 in the liver were observed at either dose.

[0442] Example 7: Antisense inhibition of MALAT1 and FOXO1 using GLP-1 peptide-conjugated antisense oligonucleotides in HEK293 cells overexpressing the human GLP-1 receptor Antisense oligonucleotides designed to target MALAT1 and FOXO1 were conjugated to glucagon-like peptide 1 receptor peptide agonists (GLP-1 peptides) 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).

[0443] The hGLP1R-HEK cell line was generated by expressing hGLP1R in Flp-IN™ 293 cells. Cultured hGLP1R-HEK cells were seeded into 96-well plates at a density of 30,000 cells / well and treated for approximately 24 hours with saline, 100 nM, or 10 μM of the unconjugated parent antisense oligonucleotide ISIS 556089 targeting MALAT1 or ISIS 776102 targeting FOXO1 (nucleobase sequence: TCTTCTTAAAATACCC) (SEQ ID NO: 64), or the corresponding GLP-1 peptide-conjugated antisense oligonucleotide (ISIS 816385 targeting MALAT1 or ION 913193 targeting FOXO1). After the treatment period, cells were harvested, mRNA was isolated, and measured using a nanadrop UV-Vis spectrophotometer and adjusted for total RNA content. MALAT1 or FOXO1 mRNA levels were measured by quantitative real-time PCR and normalized to the mRNA level of a 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 level of the housekeeping gene RPLP0 was measured using a primer-probe set with the forward sequence CCATTCTATCATCAACGGGTACAA (SEQ ID NO: 66) and the reverse sequence AGCAAGTGGGAAGGTGTAATCC (SEQ ID NO: 67).

[0444] Data are presented as percent inhibition of MALAT1 or FOXO1 mRNA relative to untreated control cells. Open symbols represent treatment with the parent antisense oligonucleotide, while closed symbols represent treatment with the antisense oligonucleotide conjugated to the GLP-1 peptide. As shown in Figure 1, in the hGLP1R-HEK cell line, the antisense oligonucleotide conjugated to the GLP-1 peptide more potently inhibited MALAT1 or FOXO1 mRNA than the parent antisense oligonucleotide.

[0445] Example 8: Dose-dependent antisense inhibition of MALAT1 following treatment with unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides in HEK293 cells overexpressing wild-type and human GPR40 or GLP-1 receptors The MALAT1 antisense oligonucleotides of Example 7 were further tested in wild-type, hGPR40 and hGLP1R-HEK cells at various concentrations.

[0446] Cultured hGLP1R-HEK, wild-type HEK293 (WT HEK293) cells, or cells expressing the hGPR40 receptor were seeded into 96-well plates at a density of 30,000 cells / well and treated with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μM antisense oligonucleotides for approximately 24 hours (concentrations are shown in Figure 2). After the treatment period, cells were harvested, mRNA was isolated, and MALAT1 mRNA levels were measured by quantitative real-time PCR using the primer probe set described herein (Example 7). Data are presented as MALAT1 mRNA levels normalized to the housekeeping gene (RPLP0). Open symbols represent treatment with the parent antisense oligonucleotide targeting MALAT1 (ISIS 556089), while closed symbols represent treatment with the same antisense oligonucleotide conjugated to a GLP-1 peptide (ISIS 816385).

[0447] The half maximal inhibitory concentration (IC50) of each oligonucleotide is shown in the table below.

[0448] [Table 1]

[0449] When conjugated to a GLP-1 peptide agonist, the antisense oligonucleotide inhibited MALAT1 gene expression 40-fold more potently in the hGLP1R-HEK cell line (Fig. 2A), but not in the WT HEK293 (Fig. 2B) or hGPR40-HEK cell lines (Fig. 2C).

[0450] Example 9: Antisense inhibition of MALAT1 and FOXO1 in mouse primary islets of Langerhans following treatment with unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides Antisense oligonucleotides targeting MALAT1 and FOXO1 were further tested in mouse primary islets of Langerhans for their ability to reduce gene expression.

[0451] Pancreatic islets were isolated by collagenase digestion of pancreata harvested from exsanguinated 12- to 15-week-old female C57BL / 6Crl mice. The islets were maintained in tissue culture until use. Pancreatic islets were dissociated into single cells by shaking in medium containing low extracellular calcium concentrations. Ten to twenty intact or dissociated islets were seeded onto plastic Petri dishes and treated with 10 μM antisense oligonucleotides for approximately 24 hours. After the treatment period, cells were harvested, and RNA was isolated and adjusted for total RNA content as measured by RIBOGREEN®. MALAT1 or FOXO1 mRNA levels were measured by quantitative real-time PCR. Mouse Malat1 mRNA levels were measured using Applied Biosystems gene expression assay Mm01227912_s1, while mouse FOXO1 mRNA levels were measured using a primer-probe set with the forward sequence CAGTCACATACGGCCAATCC (SEQ ID NO: 68), reverse sequence CGTAACTTGATTTGCTGTCCTGAA (SEQ ID NO: 69), and probe sequence TGAGCCTTTGCCCCAGATGCCTAT (SEQ ID NO: 70). All data were normalized to the mRNA level of the housekeeping gene (RPLP0) in the same sample, measured using a primer-probe set with the forward sequence GAGGAATCAGATGAGGATATGGGA (SEQ ID NO: 71), reverse sequence AAGCAGCTGACTTGGTTGC (SEQ ID NO: 72), and probe sequence TCGGTCTCTTCGACTAATCCCGCCAA (SEQ ID NO: 73).

[0452] Data as levels of MALAT1 or FOXO1 mRNA relative to the housekeeping gene (RPLP0) are shown in Figure 3. Stars represent no treatment for MALAT1 (ISIS 816385) or FOXO1 (ISIS 919193), open circles represent treatment with the parent unconjugated antisense oligonucleotide (ISIS 556089 targeting MALAT1 or ISIS 776102 targeting FOXO1), open squares represent treatment with a scrambled FOXO1 antisense oligonucleotide sequence conjugated to a GLP1 peptide (ION 913195), while closed symbols represent treatment with a GLP1 peptide-conjugated antisense oligonucleotide.

[0453] Example 10: Antisense inhibition of FOXO1 and Foxo1 protein reduction in mouse primary islets of Langerhans following treatment with unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides Antisense oligonucleotides targeting FOXO1 were tested for their ability to reduce protein levels in mouse primary islets of Langerhans.

[0454] Pancreatic 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 intact islets were placed in plastic Petri dishes and treated with 1 μM antisense oligonucleotide for 3 hours every 24 hours. They were harvested after a total treatment period of approximately 24, 48, or 96 hours, respectively. After the treatment period, the islets were harvested, and half of the islets were used to measure FOXO1 mRNA levels as described herein (Example 9). Half of the islets were homogenized in M-PER Protein Extraction Reagent (Thermo Scientific) containing a protease inhibitor cocktail (Complete Mini and phosphoSTOP, Roche Diagnostics). The protein content of the lysates was quantified using the BCA assay reagent (Pierce). FoxO1 protein was detected by Western blot analysis using the primary antibody C29H4 (Cell Signaling, #2880) against FoxO1. α-Tubulin was measured as a control for sample loading on the gel using a primary antibody from Sigma (#T6074). 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.

[0455] Inhibition of FOXO1 mRNA is shown as FOXO1 mRNA relative to the housekeeping gene and is expressed as a percentage of untreated cells in the table below, showing a slight reduction in mRNA with the unconjugated antisense oligonucleotide (ISIS 776102) and a greater than 70% reduction with the GLP-1 conjugated antisense oligonucleotide (ION 913193).

[0456] [Table 2]

[0457] Western blots showed a decrease in FoxO1 protein levels measured in pancreases treated with vehicle or antisense oligonucleotide 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 were set to 100% in vehicle-treated islets. In contrast, FoxO1 protein levels were 5% in GLP-1-FOXO1 ASO-treated islets.

[0458] Example 11: Uptake of antisense oligonucleotides in the pancreas in situ within the islets of Langerhans following administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 to C57BL / 6Crl mice Unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 were further tested in vivo to assess the uptake of antisense oligonucleotides into pancreatic islets following intravenous or subcutaneous administration of treatment.

[0459] Female C57BL / 6Crl mice were assigned to five treatment groups. Two groups received either vehicle (saline) or 2 μmol / kg GLP-1-conjugated antisense oligonucleotide (ISIS 816385) via tail vein injection. Three groups received either saline, 2 μmol / kg unconjugated parent antisense oligonucleotide (ISIS 556089), or 2 μmol / kg GLP-1-conjugated antisense oligonucleotide (ISIS 816385) subcutaneously twice weekly for two weeks. All animals were sacrificed approximately 72 hours after the final dose, and pancreases were harvested for ex vivo immunohistochemical analysis of antisense oligonucleotide uptake.

[0460] All tissues were fixed in 10% neutral buffered formalin for 32 hours at room temperature. After fixation, samples were dehydrated using a standard ethanol series followed by xylene and embedded in paraffin. Tissue sections were cut at 4 μm thickness, mounted on Superfrost® Plus slides, and baked at 60°C in a dry oven for 1 hour. Immunohistochemistry for antisense oligonucleotide detection was performed in a Ventana Discovery XT immunostainer (Ventana Medical System, Inc.) according to the manufacturer's recommendations; all reagents were Ventana products (Roche Diagnostics, Basel, Switzerland). Protease 1 was used as an enzyme antigen retrieval agent, and incubation was for 8 minutes. An antibody blocker was added for 4 minutes to reduce background, followed by rabbit anti-ASO 2.5 (dilution 1:5000, Ionis Pharmaceuticals) for 1 hour at 37°C. For secondary detection, OmiMap anti-rabbit HRP was incubated for 16 minutes, followed by chromogenic detection using the DISCOVERY ChromoMap DAB Kit (RUO). Slides were counterstained with hematoxylin for 4 minutes and then bluing for 4 minutes. Stained slides were analyzed under a standard brightfield microscope.

[0461] Antisense oligonucleotides were detected in the pancreatic islets of animals treated with ISIS 816385 administered either subcutaneously or intravenously. Oligonucleotides were not detected in the islets of animals treated subcutaneously with ISIS 556089.

[0462] Example 12: Antisense inhibition of MALAT1 within the islets of Langerhans in situ in the pancreas following administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice Unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides against MALAT1 were further tested in vivo to assess antisense inhibition of MALAT1 in the pancreas after intravenous or subcutaneous administration of treatment.

[0463] Female C57BL / 6Crl mice were assigned to five treatment groups as described herein (Example 11). Animals were sacrificed approximately 72 hours after the final dose, and pancreases were harvested for ex vivo analysis of MALAT1 expression by in situ hybridization.

[0464] Tissues were prepared as described herein (Example 11). The in situ mRNA amplification and labeling process was performed on a Ventana Discovery ULTRA, Automated ISH platform (Ventana Medical System, Inc.) using the Advanced Cell Diagnostics (ACD)-based RNAscope® VS assay. Custom probes were obtained from ACD for detection of MALAT1 mRNA, and various parameters were tested to optimize the novel RNAscope method for ISH. Multiple steps were used to amplify the signal, and then the probe was labeled and detected using the RNAscope® 2.5 VS Reagent Kit-RED. Stained slides were analyzed under a standard bright-field microscope.

[0465] MALAT1 expression was reduced in pancreatic islets of Langerhans in animals treated subcutaneously or intravenously with a GLP-1 peptide-conjugated antisense oligonucleotide (ISIS 816385), but not in exocrine tissues. MALAT1 expression was not reduced in animals treated subcutaneously with the unconjugated parent antisense oligonucleotide (ISIS 556089).

[0466] Example 13: Uptake of antisense oligonucleotides into the liver 72 hours after administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 to C57BL / 6Crl mice The unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides against MALAT1 were further tested in vivo to assess the uptake of antisense oligonucleotides into the liver by either intravenous or subcutaneous routes of administration.

[0467] Animals were assigned to treatment as described herein (Example 11). All animals were sacrificed approximately 72 hours after the final dose, and pancreases were harvested for ex vivo analysis by immunohistochemistry of antisense oligonucleotide uptake.

[0468] Tissues were prepared and immunohistochemistry was performed as described herein (Example 12).

[0469] Antisense oligonucleotides were detected in hepatocytes and Kupffer cells in the livers of animals treated with both ISIS 816385 and ISIS 556089 administered either subcutaneously or intravenously as indicated.

[0470] Example 14: Antisense inhibition of MALAT1 in liver following administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice Unconjugated parent (ISIS 556089) and GLP-1 peptide-conjugated antisense oligonucleotides against MALAT1 were further tested in vivo to evaluate antisense inhibition of MALAT1 in the liver by intravenous and subcutaneous administration routes.

[0471] Female C57BL / 6Crl mice were assigned to five treatment groups as described herein (Example 13). All animals were sacrificed approximately 72 hours after the end, and livers were harvested for ex vivo analysis of MALAT1 expression by in situ hybridization.

[0472] Tissues were prepared and in situ hybridization was performed as described herein (Example 12).

[0473] Hepatic MALAT1 expression was reduced in hepatic parenchymal cells of animals treated with ISIS 816385 to a greater extent than in hepatic parenchymal cells of animals treated with ISIS 556089 administered subcutaneously. Hepatic MALAT1 was also reduced compared to vehicle controls in animals administered ISIS 816385 intravenously.

[0474] Example 15: Dose-dependent antisense inhibition of MALAT1 in isolated islets and liver 72 hours after administration of a single dose of unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice The unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the efficacy of antisense inhibition of MALAT1 in isolated pancreatic islets versus liver 72 hours after a single subcutaneous administration.

[0475] Female C57BL / 6Crl mice were assigned to eight treatment groups and received a single subcutaneous injection of either vehicle, 0.01 μmol / kg, 0.03 μmol / kg, 0.1 μmol / kg, or 1 μmol / kg ISIS 816385; the other three treatment groups received 0.01 μmol / kg, 0.1 μmol / kg, or 1 μmol / kg ISIS 556089. All animals were sacrificed 72 hours after the final dose. For mRNA analysis, liver samples were collected and pancreatic islets were isolated as described herein (Example 9). MALAT1 mRNA levels were quantified as described herein (Example 9) and expressed as a percentage of vehicle-treated animals (controls).

[0476] No significant antisense inhibition of MALAT1 was observed in the liver in either treatment group or in the islets of Langerhans from animals treated with the parent antisense oligonucleotide (ISIS 556089). The GLP-1 peptide-conjugated antisense oligonucleotide dose-dependently inhibited MALAT1 mRNA levels with an estimated ED50 of 0.07 μmol / kg.

[0477] Example 16: Dose-dependent antisense inhibition of FOXO1 in isolated islets and liver 72 hours after administration of a single dose of unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice The unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the efficacy of antisense inhibition of FOXO1 in isolated pancreatic islets versus liver 72 hours after a single dose subcutaneous administration.

[0478] Female C57BL / 6Crl mice were assigned to seven treatment groups and received a single subcutaneous injection of either vehicle, 0.01 μmol / kg, 0.03 μmol / kg, 0.1 μmol / kg, or 1 μmol / kg ION 913193; two treatment groups received 0.01 μmol / kg or 1 μmol / kg ISIS 776102. All animals were sacrificed 72 hours after the final dose. Liver samples were collected and pancreatic islets were isolated for mRNA analysis as described herein (Example 9). FOXO1 mRNA levels were quantified as described herein (Example 9) and expressed as a percentage of vehicle-treated animals (controls).

[0479] No significant antisense inhibition of FOXO1 in the liver or islets of Langerhans was observed in either treatment group treated with the parent antisense oligonucleotide (ISIS 776102). The GLP-1 peptide-conjugated antisense oligonucleotide dose-dependently inhibited FOXO1 mRNA levels with an estimated ED50 of 0.04 μmol / kg.

[0480] Example 17: Antisense inhibition of FOXO1 in isolated islets and liver 6 weeks after repeated administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides to ob / ob mice The unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to assess the efficacy of antisense inhibition of FOXO1 in isolated pancreatic islets versus liver after 6 weeks of treatment.

[0481] Male ob / ob mice (B6.V-Lepob / OlaHsd, Harlan) were assigned to five treatment groups and received either vehicle, 0.1 μmol / kg ISIS 776102, 0.1 μmol / kg ION 913195, 0.03 μmol / kg ION 913193, or 1 μmol / kg ION 913193. All animals were treated weekly for 6 weeks. Approximately 120 hours after the final dose, all animals were sacrificed, and liver samples were collected and pancreatic islets were isolated for mRNA analysis as described herein (Example 9). FOXO1 mRNA levels were quantified as described herein (Example 9), and mRNA levels were normalized to the housekeeping gene (RPLP0) in each sample.

[0482] No significant antisense inhibition of FOXO1 was observed in the liver of any of the treatment groups or in islets treated with the parent antisense oligonucleotide (ISIS 776102) or a scrambled FOXO1 antisense oligonucleotide sequence conjugated to a GLP-1 peptide (ION 913195). GLP-1 peptide-conjugated antisense oligonucleotide (ION 913193)-treated animals reduced FOXO1 mRNA levels in isolated islets at both dose levels tested (42% mean FOXO1 mRNA reduction at 0.03 μmol / kg and 72% mean FOXO1 mRNA reduction at 0.1 μmol / kg), demonstrating that GLP-1 peptide conjugation improves antisense inhibition in pancreatic islets in vivo.

[0483] Example 18: Reduction of FoxO1 protein levels in islets isolated from ob / ob mice treated with unconjugated parental or GLP-1 peptide-conjugated antisense oligonucleotides for 6 weeks The unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides were further tested for their ability to reduce FoxO1 protein levels in mouse pancreatic islets of Langerhans isolated from ob / ob mice treated for 6 weeks.

[0484] Male ob / ob mice were assigned to five treatment groups as described herein (Example 17). Approximately 120 hours after the final dose, all animals were sacrificed, and pancreatic 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, ensuring that at least one sample from each treatment group was analyzed on the same gel. FoxO1 protein levels were measured by quantifying intensity and normalized to α-tubulin levels in the same sample. All samples in each gel were expressed as a percentage of the levels measured in the pancreatic islets of animals that received ION 913195.

[0485] Foxo1 protein levels were reduced by 57% and 81% in animals treated with ION 913193 versus ION 913195 at 0.03 μmol / kg and 0.1 μmol / kg, respectively, and by 64% and 36% versus 0.1 μmol / kg ISIS 776102.

[0486] Example 19: Preparation of GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 ION 962963, a 5′-GLP-1 peptide-conjugated ASO targeting MALAT1, was prepared according to the procedure of Example 1, starting from a 5′-hexylamino-modified oligonucleotide (ISIS 722061) (nucleobase sequence: GCATTCTAATAGCAGC (SEQ ID NO: 65)) with the chemical modifications Gks mCks Aks Tds Tds mCds Tds Ads Ads Tds Ads Gds mCds Aks Gks mCk (k=cEt; d=2′-deoxy; e=2′-MOE; mC=5-methylcytosine; o=phosphodiester; and s=phosphorothioate).

[0487] Example 20: Preparation of antisense oligonucleotides targeting MALAT1 conjugated to GLP-1 peptides via click linkers A method for preparing a conjugated modified oligonucleotide comprising GLP-1 conjugated at the 5' position via a click linker.

[0488] Preparation of 5'-BCN MALAT-1 targeting oligonucleotide ISIS 791173: 5'-Hexylamino-modified oligonucleotide (ISIS 786434) (nucleobase sequence: TCAGCATTCTAATAGCAGC (SEQ ID NO: 58)) was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5' end of the modified oligonucleotide contains a hexamethylene linker and a terminal amine. BCN-NHS ester (Mol. Wt 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 and added to the ASO solution. The mixture was 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.

[0489] Preparation of GLP-1 click-conjugated ASO (ION 1071996) Twelve mg of the modified oligonucleotide ISIS 791173 was dissolved in 1 mL of 0.1 M sodium tetraborate, pH 8.5 (ASO solution), and 12 mg of N-terminal azido-GLP-1 peptide was dissolved in 400 μL of DMF (peptide solution). The peptide solution was added to the ASO solution and stirred at room temperature for 18 hours. At the 18-hour point, precipitation was observed, and 1 mL of additional DMF was added. The reaction was continued for an additional 5 hours. The product was purified by HPLC on a SAX column using Buffer A 100 mM NHOAc / 30% ACN / H2O and Buffer B 1.5 M NaBr / NHOAc / 30% ACN / H2O and desalted by HPLC on a reverse-phase column. The product fractions were collected and lyophilized to yield the expected conjugated ASO, ION 1071996. [ka]

[0490] Example 21: Antisense inhibition of MALAT1 in mouse primary islets of Langerhans after treatment with unconjugated parent or GLP-1 peptide-conjugated ASOs with various linkers To determine whether the chemical structure of the GLP-1 peptide conjugation to the ASO affects ASO delivery into the pancreas, male C57BL / 6 mice received weekly intravenous injections of 0.6 μmol / kg / week of vehicle (saline), ISIS 556089 (parent 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) for 3 weeks. Tissues were collected 72 hours after the final injection to assess compound delivery and efficacy.

[0491] MALAT1 expression was detected as in Example 6. In situ hybridization analysis showed that MALAT1 expression in beta islet cells was reduced 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.

[0492] Example 22: Dose-dependent reduction of MALAT-1 expression in LVX-GLP1R cells HEK cells stably expressing FLAG-tagged GLP1R were generated by transfection of 293T cells with pLVX-IRES-Puro (Clontech Laboratories Inc., Mountainview, CA) containing a FLAG-GLP1R insert and then infected with FLAG-tagged GLP1R-containing lentivirus. Infected cells were selected with puromycin (2 μg / ml) and then analyzed for receptor expression by Western blot and immunofluorescence. Cultured GLP1R cells were seeded at a density of 10,000 cells / 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 an 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 total RNA in 5 μL water was mixed in a 20 μL reaction with 0.3 μL primer-probe set containing forward and reverse primers (10 μM each), a fluorescently labeled probe (3 μM), 0.3 μL room temperature enzyme mix (Qiagen), 4.4 μL RNase-free water, and 10 μL of 2× PCR reaction buffer. PCR was performed using a StepOne Plus RT-PCR system (Applied Reverse transcription was performed at 48°C for 10 min using a PCR kit (Life Technologies, Phoenix, AZ, USA), followed by 40 cycles of PCR with 94°C for 20 s and 60°C for 20 s during each cycle. mRNA levels were normalized to the amount of total RNA present in each reaction as determined by Ribogreen assay (Life Technologies) and normalized to a saline control (100% expression).The results are shown in the table below and demonstrate increased dose-dependent inhibition of MALAT-1 by GLP-1 conjugated ASO with (816385) or without (962963) a TCA linker.

[0493] [Table 3]

[0494] Example 23: Effect of peptide length and conjugation position on the in vitro activity of GLP-1-conjugated ASOs targeting MALAT1 To assess the effects of precise peptide sequence, peptide length, and conjugation position on the in vitro activity of GLP-1-conjugated ASOs complementary to MALAT1, a series of modified oligonucleotides with variations in peptide sequence were synthesized by click chemistry. All peptides have a C-terminal amide. ION 1083582 was synthesized from ION 791173 by click reaction with a 5-azidopentanoic acid-modified lysine residue (X) as shown below. Other compounds were synthesized from ION 791173 by click reaction with a C-terminal azidonorleucine (Z), as shown in Example 20 above. [ka]

[0495] LVX-GLP1R cells (described in Example 22) were seeded at a density of 10,000 cells / well and incubated with a 4-fold dilution series of seven doses of peptide-conjugated ASO. After the treatment period, total RNA was prepared and analyzed as in Example 22 above. IC50s are shown in the table below.

[0496] [Table 4]

[0497] Example 24: Preparation of GLP-1 conjugated siRNA targeting PTEN Methods for preparing PTEN-targeting siRNA nucleotide duplexes ISIS 522247 (nucleobase sequence TTATCTATAATGATCAGGTAA (SEQ ID NO: 77) and chemically modified Afs Cms Cfo Umo Gfo Amo Ufo Cmo Afo Umo Ufo Amo Ufo Amo Gfo Amo Ufs Ams Af (as above)) with the chemical modifications Txs Ufs Amo Ufs Cmo Ufs Amo Ufs Amo Afs Umo Gfs Amo Ufs Cms Afs Gms Gfs Ums Aes Ae (Tx=5'-(E)-vinylP-2'-O-methoxyethyl-thymine, f=2'-α-fluoro-2'deoxyribose, m=2'-O-methylribose, e=2'O-methoxyethylribose, o=phosphodiester; and s=phosphorothioate) 790973 (nucleobase sequence ACCTGATCATTATAGATAA (SEQ ID NO: 78) was synthesized and purified using standard solid phase oligonucleotide procedures.

[0498] 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 Cms Cfo 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 HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGlyProSerSerGlyAlaProProProSerCys, which contains a free N-terminal amine and C-terminal amide.

[0499] ISIS 522247 was hybridized with 790973 to form a duplex of the two oligonucleotides. ISIS 522247 was hybridized with 1055394 to form a duplex of the two oligonucleotides.

[0500] Example 25: Preparation of GLP-1 antagonist-conjugated oligonucleotides Method for the synthesis of GLP-1 antagonist conjugated oligonucleotide, DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC-SS-propionyl-HA-o-TdomCdoAdoGksmCksAks TdsTdsmCdsTdsAdsAdsTdsAdsGdsmCdsAksGksmCk (ION 998975).

[0501] 38 mg of the linker-ISIS 786434 (compound 2) described in Example 1 was dissolved in 1.5 mL H2O, and 0.5 mL of 0.1 M NaHCO3 / H2O was added to adjust the pH to about 7.5 to 8.0 (ASO solution).

[0502] 27.7 mg of the 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 min. The reaction was monitored by LCMS, and stirring was continued for an additional 1 h. The major fraction was found to be the expected product. The product was diluted with water and kept at 4 °C until purified by HPLC on a strong anion exchange column (Buffer A = 100 mM ammonium acetate in 30% acetonitrile in water; Buffer B = 1.5 M NaBr in A, 0-60% B in 28 column volumes). Fractions containing the full-length ASO were pooled together, diluted to a 10% concentration in acetonitrile, 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 give the expected product which was confirmed by LCMS.

[0503] Example 26: Method for preparing a conjugated modified oligonucleotide containing GLP-1 conjugated at the 5' position via a maleimide linker. A 5'-hexylamino-modified oligonucleotide (ISIS 786434) targeting MALAT1 was synthesized and purified as previously described herein. ISIS 786434 was reacted with 5 eq. of N-succinimidyl 3-maleimidopropionate (MW 266.21 g / mol) in sodium tetraborate buffer at pH 7 and room temperature to yield 5'-(3-maleimido)propionyl-C6 MALAT1 ASO. A GLP-1 peptide containing a C-terminal cysteine ​​amide ("GLP-1 peptide-cysteine ​​amide", HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2) was dissolved in 0.1 M sodium phosphate, pH 8.5 / DMF, and added to the 5'-(3-maleimido)propionyl-C6 MALAT1 ASO with stirring at room temperature. The product (ION1086699) was formed.

[0504] Example 27: Method for preparing a conjugated modified oligonucleotide containing GLP-1 conjugated at the 5' position via a disulfide-click linker (preparation of Ionis-1123478). A 5'-hexylamino-modified oligonucleotide (ISIS 786434) targeting MALAT1 was synthesized on a NittoPhase® HL solid support (115 mg, 47 μmol) using an AKTA Oligopilot™ synthesizer. A 0.1 M solution of the thiol modifier C6 SS amidite (Glen research 10-1936-02, 0.25 g in 3.2525 mL of dry ACN) and N-(4-monomethoxytrityl (MMT))-6-amino-1-hexanol amidite (0.177 g in 3 mL of dry ACN) was coupled to a solid support bearing Ion 925727 using standard solid-phase oligonucleotide procedures. The 5'-MMT-protected oligonucleotide was cleaved, precipitated, and purified as previously described herein. The MMT-protected oligonucleotide was dissolved in 50 mL of water and 50 mL of 3 M sodium acetate solution (pH 5.0) and heated at 45°C for 60 min. The reaction mixture was cooled from 45 to 22°C, and the pH was raised to 5.92 by adding 10% v / v 2.0 M buffered sodium acetate solution (pH 7.2). The reaction was considered stopped upon completion of sodium acetate addition. The product was purified by HPLC on Source 30Q resin using Buffer A 100 mM NHOAc / 30% ACN / H2O and Buffer B 100 mM NHOAc / 30% ACN / H2O + 1.5 M NaBr. Pure fractions were desalted on a reverse-phase column by HPLC to yield the MMT-deprotected oligonucleotide.

[0505] The deprotected oligonucleotide was then reacted with 3 eq. of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (Aldrich 744867) in 1:2 DMSO-sodium tetraborate buffer, pH 8.5, at room temperature to yield the 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 a C-terminal 4-AzidoNorLeu [GLP1 / Ex4 Fusion Sequence-40 N3-NH2, H2N-HAibEGTFTSDVSSYLE EQAAKEFIAW LVKGGPSSGAPPPS(4-AzidoNorLeu)-NH2] was dissolved in 1 mL of DMSO and added to the ASO solution. After 3 h, the reaction mixture was diluted with water (5x the volume of the reaction solution, v / v), and the product was purified by HPLC on Source 30Q resin using Buffer A 100 mM NHOAc / 30% ACN / HO and Buffer B 100 mM NHOAc / 30% ACN / HO + 1.5 M NaBr. The product fractions were desalted on reverse-phase HPLC to give ION 1123478, which was confirmed by LC-MS analysis. [ka]

[0506] Example 28: Method for preparing a conjugated modified oligonucleotide containing GLP-1 conjugated at the 5' position via a maleimide acid linker (Preparation of Ionis-1123118). A 5'-hexylamino-modified oligonucleotide (ISIS 786434) targeting MALAT1 was synthesized and purified as previously described herein. ISIS 786434 was reacted with 5 eq. of N-succinimidyl 3-maleimidopropionate (MW 266.21 g / mol) in sodium tetraborate buffer at pH 7 at room temperature to yield a 5'-(3-maleimidyl)propionyl-C6 MALAT1-modified oligonucleotide. The 5'-modified oligonucleotide was purified by SAX IE HPLC using a linear gradient of buffers A and B. Buffer A: 100 mM NHOAc in acetonitrile:water 3:7 (v:v), buffer B: 1.5 M NaBr, 100 mM NHOAc in acetonitrile:water 3:7 (v:v), and desalted using reverse-phase HPLC. A GLP-1 peptide containing a C-terminal cysteine ​​amide ("GLP-1 peptide-cysteine ​​amide," HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2) was dissolved in 0.1 M sodium phosphate, pH 7.0, in DMF and added to a solution of 5'-(3-maleimudyl)propionyl-C6 MALAT1-modified oligonucleotide with stirring at room temperature. The product (ION 1086699) was formed and purified by SAX IE HPLC using a linear gradient of buffers A and B. Buffer A: 50 mM NaHCO3 in 3:7 acetonitrile:water (v:v); Buffer B: 1.5 M NaBr, 50 mM NaHCO3 in 3:7 acetonitrile:water (v:v). Product fractions were pooled, kept at 5 °C for 4 days, and desalted on a reverse-phase column by HPLC to give ION 1123118, which was confirmed by LC-MS analysis. [ka]

[0507] Example 29: Antisense inhibition of MALAT1 in isolated islets of Langerhans in C57BL / 6Crl mice 72 hours after administration of a single dose of GLP-1 peptide-conjugated antisense oligonucleotides with various linkers To determine whether the chemistry of GLP-1 peptide conjugation to ASOs affects ASO delivery into the pancreas, female C57BL / 6Crl mice received a single subcutaneous injection of 0.01 μmol / kg of vehicle (saline), ISIS 816385 (GLP-1-conjugated ASOs with a disulfide linker), ION 1071996 (GLP-1-conjugated ASOs conjugated via a click linker), ION 1086699 (GLP-1-conjugated ASOs conjugated via a maleimide linker), ION 1123118 (GLP-1-conjugated ASOs conjugated via a maleimide acid linker), or ION 1123478 (GLP-1-conjugated ASOs conjugated via a disulfide-click linker). Islets were isolated 72 hours after subcutaneous administration to assess compound delivery and efficacy.

[0508] MALAT1 expression was measured as in Example 9, expressed as a percentage of vehicle-treated animals (control), and compared to expression in islets from mice treated with ISIS 816395. qPCR quantification shows that MALAT1 expression in β-islet cells was significantly further reduced in mice treated with the GLP-1-conjugated ASOs ION 1086699, ION 1123118, and ION 1123478, and similarly reduced in mice treated with ION 1071996, compared to ISIS 816395.

[0509] [Table 5]

[0510] Example 30: Dose-responsive antisense inhibition of MALAT1 in isolated islets of Langerhans 72 hours after administration of a single dose of GLP-1 conjugated antisense oligonucleotides with various linkers to C57BL / 6Crl mice To determine whether the chemistry of conjugation of the GLP-1 peptide to the ASO affects ASO delivery into the pancreas, female C57BL / 6Crl mice (n=6) received a single subcutaneous injection of 0.03, 0.01, 0.001, or 0.0003 μmol / kg 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 maleimide acid linker), or ION 1134165 (GLP-1-conjugated MALAT-1 ASO conjugated via a disulfide linker). Groups of mice (n=6) were subcutaneously injected with 0.01 μmol / kg ISIS 816385 (GLP-1-conjugated MALAT-1 ASO conjugated via a disulfide linker, as described in Example 1). Groups of mice (n=6) were subcutaneously injected with PBS, which served as a control group to which other groups were compared. Islets were isolated 72 hours after subcutaneous administration to assess compound delivery and efficacy. Percent inhibition of MALAT1 mRNA levels was quantified as described herein (Example 9) and averaged for each treatment group relative to PBS-treated animals (control). 0% inhibition reflects no observed inhibition of MALAT1 mRNA. ND means that no mice received the indicated dose.

[0511] [Table 6]

[0512] The results show that GLP-1-conjugated MALAT-1 ASOs conjugated via a maleimide acid linker showed a higher dose-response inhibition of MALAT-1 mRNA in pancreatic islet cells compared to GLP-1-conjugated MALAT-1 ASOs conjugated via a maleimide or disulfide linker.

[0513] Example 31: Effect of linkers on food intake in C57BL / 6Crl mice treated with GLP-1 conjugated antisense oligonucleotides Groups of eight mice were administered a single subcutaneous injection of 0.01, 0.03, or 0.1 μmol / kg ION 816385 (GLP-1-conjugated MALAT-1 ASO conjugated via a disulfide linker) or 0.1 μmol / kg ION 1123118 (GLP-1-conjugated MALAT-1 ASO conjugated via a maleimide acid linker) as shown in the table below, and food intake was monitored for 24 hours. Food intake of eight mice administered a subcutaneous injection of PBS was monitored as a control. The maleimide acid linker increased the therapeutic window associated with GLP-1-induced food intake reduction compared to the disulfide linker.

[0514] [Table 7] The present application also includes the following aspects. [Aspect 1] 1. A compound comprising an oligonucleotide linked by a conjugate linker to a GLP-1 receptor ligand conjugate moiety, [ka] wherein N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly 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 said oligonucleotide A compound comprising: [Aspect 2] 1. A compound comprising an oligonucleotide linked by a conjugate linker to a GLP-1 receptor ligand conjugate moiety, [ka] wherein N-N=N represents an azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly 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 said oligonucleotide A compound comprising: [Aspect 3] In certain embodiments, the compound comprises an oligonucleotide linked to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, said compound comprising:

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Claims

1. 1. A compound comprising an oligonucleotide attached to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, said conjugate linker comprising: (A) 【Chemistry 1】 (Wherein R is (CH ２ ) ｎ and n is 1 to 12; or R is 【Chemistry 2】 and m is 1 to 12; (B) 【Transformation 3】 and (C) 【Chemistry 4】 (where X is directly or indirectly attached to said GLP-1 receptor ligand conjugate moiety; and Y is attached directly or indirectly to said oligonucleotide), the GLP-1 receptor ligand conjugate moiety is a GLP-1 peptide conjugate moiety that targets the GLP-1 receptor; the GLP-1 peptide conjugate moiety comprises an amino acid sequence of any of SEQ ID NOs: 2-21 and 24-57; compound.

2. 2. The compound of claim 1, wherein the oligonucleotide is a modified oligonucleotide of 12 to 30 linked nucleosides in length.

3. The compound of claim 2 , wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar, or at least one modified nucleobase.

4. the modified oligonucleotide is a gap segment consisting of linked deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and 3' wing segment consisting of linked nucleosides wherein the gap segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar.

5. The compound of any one of claims 2 to 4, wherein the modified oligonucleotide is single-stranded.

6. The compound of any one of claims 2 to 5, wherein the modified oligonucleotide is complementary to an RNA transcript in a pancreatic beta islet cell.

7. The compound of claim 6 , wherein the RNA transcript is a pre-mRNA, mRNA, non-coding RNA, or miRNA.

8. The compound according to any one of claims 1 to 7, wherein the GLP-1 peptide conjugate moiety consists of an amino acid sequence of any one of SEQ ID NOs: 2 to 21 and 24 to 57.

9. The compound of any one of claims 1 to 8, wherein the GLP-1 peptide conjugate moiety binds to a GLP-1 receptor expressed on the surface of pancreatic beta islet cells.

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