GLP-1 receptor ligand partially conjugated oligonucleotide and its use

Abstract

This invention provides compounds and methods for targeting cells expressing the GLP-1 receptor. [Solution] A compound comprising an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, JPEG2026094156000137.jpg33161 (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the remaining portion of the GLP-1 receptor ligand conjugate moiety; n and o are independently selected from 2 to 10; and Y is directly or indirectly attached to the oligonucleotide.) A compound containing the following is provided.

Description

[Technical Field]

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

[0002] This embodiment provides 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 binds to adenylyl cyclase via a stimulating G protein receptor. Intestinal nutritional stimulation releases glucagon-like peptide-1 into circulation. Circulating GLP-1 binds to GLP-1 receptors on β-islet cells of the pancreas. This activates the GLP-1 receptor, inducing a signaling event that leads to insulin exocytosis from the β-islet cells. The binding of GLP-1 and the GLP-1 receptor causes the receptor to translocate into the cytoplasm and ultimately be sorted into lysosomes (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

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

[0005] Embodiments provided herein relate to compounds and methods for modulating the expression of nucleic acid targets in cells expressing the 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, contact of cells expressing the GLP-1 receptor, such as pancreatic β-islet cells, with the compounds provided herein modulates the expression of nucleic acid targets in the cells. In certain embodiments, the compound comprising the GLP-1 receptor ligand conjugate moiety selectively or preferentially targets cells expressing the GLP-1 receptor compared to cells not expressing the GLP-1 receptor. In certain embodiments, the compound comprising the GLP-1 receptor ligand conjugate moiety selectively or preferentially targets cells expressing the GLP-1 receptor compared to compounds not comprising the GLP-1 receptor ligand conjugate moiety. [Brief explanation of the drawing]

[0006] [Figure 1] These graphs show the percentage of FOXO1 mRNA (Figure 1A) and MALAT1 mRNA (Figure 1B) relative to the antisense oligonucleotide (ASO) concentration in HEK293 cells treated with either a non-conjugated parent ASO (ISIS 776102 or ISIS 556089) or a GLP1-conjugated ASO (ISIS 913193 or ISIS 816385). [Figure 2] This graph shows the MALAT1 mRNA levels in relation to antisense oligonucleotide (ASO) concentrations in GLP1 receptor-overexpressing HEK293 cells (Figure 2A), wild-type HEK293 cells (Figure 2B), or GRP40-overexpressing HEK293 cells (Figure 2C) treated with unconjugated parental MALAT1 ASO (ISIS 556089) or GLP1-conjugated MALAT1 ASO (ISIS 816385). [Figure 3]MALAT1 mRNA levels in dispersed mouse pancreatic 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 pancreatic 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 pancreatic 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) This is a graph showing mRNA levels (Figure 3C). [Modes for carrying out the invention]

[0007] It should be understood that both the summary above and the detailed description below are merely illustrative and descriptive and do not limit the claimed embodiments. In this specification, a singular use includes the plural unless otherwise stated. In this specification, a use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “contains” and other forms such as “contains” and “includes” is not limiting.

[0008] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. All or part of the documents cited herein, including, but not limited to, patents, patent applications, articles, books, papers, and GenBank and NCBI reference sequencing records, are expressly incorporated herein by reference, both in part and in whole, into this specification.

[0009] It is understood that the sequences shown in each sequence number of the example oligonucleotides included herein are independent of any modifications to the sugar moiety, nucleoside bond, or nucleic acid base. Therefore, an oligonucleotide defined by a sequence number may independently contain one or more modifications to the sugar moiety, nucleoside bond, or nucleic acid base. Oligonucleotides described by an ISIS or ION number (ISIS# or ION#) represent a combination of nucleic acid base sequence, chemical modification, and motif.

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

[0011] Unless otherwise specified, the following terms have the following meanings:

[0012] "2'-deoxynucleoside" refers to a nucleoside containing a 2'-H(H) furanosyl sugar moiety found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleic acid base or an RNA nucleic acid base (uracil).

[0013] "2'-O-methoxyethyl" (also known as 2'-MOE and 2'-O(CH2)2-OCH3) refers to the O-methoxyethyl modification at the 2' position of the furanosyl ring. 2'-O-methoxyethyl modified sugars are modified sugars.

[0014] "2'-MOE nucleoside" (also called 2'-O-methoxyethyl nucleoside) refers to a nucleoside that contains a 2'-MOE modified sugar moiety.

[0015] "2'-substituted nucleoside" or "2-modified nucleoside" means a nucleoside containing 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 containing at least one 2'-substituent other than H or OH.

[0016] "5-methylcytosine" refers to cytosine that has a methyl group attached at the 5th position.

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

[0018] "Administration" or "administering" refers to a route through which a compound or composition provided herein is introduced into an individual to perform its intended function. Examples of usable routes of administration include, but are not limited to, parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

[0019] "Aminoisobutyric acid" or "Aib" is the formula unless otherwise specified. [ka] This refers to 2-aminoisobutyric acid, which contains [the specified ingredient].

[0020] "Animals" refers to humans or non-human animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[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 reduction in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid, compared to the level of the target nucleic acid or target protein in the absence of the antisense compound against the target.

[0022] An "antisense compound" refers to a compound comprising an oligonucleotide and optionally one or more additional feature groups, such as a conjugate group or 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" refers to the reduction of the target nucleic acid level in the presence of an antisense compound complementary to the target nucleic acid, compared to the target nucleic acid level in the absence of the antisense compound.

[0024] "Antisense mechanisms" include all mechanisms, including hybridization of a compound with a target nucleic acid, in which the outcome or effect of hybridization is either targeted degradation or targeted occupation, for example, in which cellular mechanisms, including transcription or splicing, are incidentally stalled.

[0025] "Antisense oligonucleotide" means an oligonucleotide having a nucleic acid base sequence complementary to a target nucleic acid or a region or segment thereof. In certain embodiments, the antisense oligonucleotide can specifically hybridize to a target nucleic acid or a region or segment thereof.

[0026] "Bicyclic nucleoside" or "BNA" means a nucleoside containing a bicyclic sugar moiety. "Bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety containing two rings (the second ring is formed via a bridge connecting two atoms in the first ring, thereby forming a bicyclic structure). In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not contain a furanosyl moiety.

[0027] A "branched chain 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 chain group provides multiple reactive sites for linking a tethered ligand to an oligonucleotide via a conjugate linker and / or cleavable portion.

[0028] The "cell-targeting moiety" means a conjugate group or a portion of a conjugate group that can bind to one or more specific cell types.

[0029] "cEt" or "restricted ethyl" refers to a bicyclic furanosyl sugar moiety containing a bridge linking the 4'-carbon and 2'-carbon atoms (the bridge has the formula: 4'-CH(CH3)-O-2').

[0030] "Chemical modification" in a compound describes a substitution or change of any unit in that compound via a chemical reaction. "Modified nucleoside" refers to a nucleoside independently possessing a modified sugar moiety and / or a modified nucleic acid base. "Modified oligonucleotide" refers to an oligonucleotide containing at least one modified nucleoside bond, a modified sugar, and / or a modified nucleic acid base.

[0031] A "chemically distinct region" refers to a region of a compound that is chemically different from another region of the same compound. For example, a region containing a 2'-O-methoxyethyl nucleotide is chemically distinct from a region containing a nucleotide that does not have the 2'-O-methoxyethyl modification.

[0032] A "chimeric antisense compound" refers to an antisense compound having at least two chemically distinct regions, each having multiple subunits.

[0033] A "cleavable bond" means any chemical bond that can be divided. In certain embodiments, the cleavable bond is selected from amides, polyamides, esters, ethers, one or both phosphodiesters, phosphate esters, carbamates, disulfides, or peptides.

[0034] "Crackable portion" means a bond or group of atoms that can be cleaved under physiological conditions, for example, within a cell, animal, or human.

[0035] With respect to oligonucleotides, "complementary" means that the nucleic acid base sequence of such oligonucleotide or one or more regions thereof matches the nucleic acid base sequence of another oligonucleotide or nucleic acid or one or more regions thereof when these two nucleic acid base sequences are aligned in the opposite direction. Unless otherwise specified, the nucleic acid base matches or complementary nucleic acid bases described herein are the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine ( m Limited to C) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleic acid base complementarity at each nucleoside and may contain one or more nucleic acid base mismatches. In contrast, “fully complementary” or “100% complementary” with respect to oligonucleotides means that such oligonucleotides have nucleic acid base matches at each nucleoside without any nucleic acid base mismatches.

[0036] A "conjugate group" refers to an atomic group attached to an oligonucleotide. A conjugate group includes a conjugate portion and a conjugate linker that attaches the conjugate portion to the oligonucleotide.

[0037] A "conjugate linker" refers to a group of atoms that contains at least one bond that links the conjugate portion to an oligonucleotide.

[0038] The "conjugate portion" refers to the group of atoms attached to the oligonucleotide via a conjugate linker.

[0039] "To design" or "to be designed to do something" refers to the process of designing a compound that specifically hybridizes with a selected nucleic acid molecule.

[0040] "Differentially modified" means having different chemical modifications or substituents, including the absence of modifications. Therefore, 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 nucleic acid bases are not differentially modified. For example, a nucleoside containing a 2'-OMe modified sugar and an unmodified adenine nucleic acid base is not differentially modified, as is a nucleoside containing a 2'-OMe modified sugar and an unmodified thymine nucleic acid base.

[0041] A "double-stranded antisense compound" is an antisense compound comprising two complementary oligomeric compounds that form a double chain, wherein one of the two oligomeric compounds is an antisense compound containing an oligonucleotide.

[0042] "Expression" encompasses all functions in which the coding information of a gene is converted into structures that exist and function within a cell. Such structures include, but are not limited to, the products of transcription and translation.

[0043] A "gapmer" refers to an oligonucleotide containing an internal region with multiple nucleosides that support RNase H cleavage, located between external regions having one or more nucleosides. The nucleosides containing the internal region are chemically distinct from the nucleosides or nucleotides containing the external region. The internal region can be called a "gap," and the external region can be called a "wing."

[0044] "Hybridization" refers to the annealing of oligonucleotides and / or nucleic acids. While not limited to specific mechanisms, the most common mechanisms of hybridization involve hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds 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 decrease or interference with the expression or activity compared to the untreated or control sample, and does not necessarily mean the complete elimination of expression or activity.

[0046] "Nucleotide interbonding" refers to the group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. "Modified nucleoside interbonding" refers to any nucleoside interbonding other than naturally occurring phosphate nucleoside interbonding. Non-phosphate bonds are referred to herein as modified nucleoside interbonding.

[0047] "Bound nucleoside" refers to an adjacent nucleoside that is bound together by an internucleoside bond.

[0048] A "linker nucleoside" refers to a nucleoside that binds an oligonucleotide to the conjugate portion of a compound. The linker nucleoside is located within the conjugate linker of the compound. Even if it is contiguous with the oligonucleotide, the linker nucleoside is not considered part of the oligonucleotide portion of the compound.

[0049] "Mismatched" or "non-complementary" means that, when the first and second oligonucleotides are aligned, the nucleic acid base of the first oligonucleotide is not complementary to the nucleic acid base of the second oligonucleotide or the corresponding nucleic acid base of the target nucleic acid. For example, nucleic acid bases, such as, but not limited to, universal nucleic acid bases, inosine, and hypoxanthine, can hybridize with at least one nucleic acid base, but are still mismatched or non-complementary to the nucleic acid base they hybridize with. As another example, when the first and second oligonucleotides are aligned, the nucleic acid base of the first oligonucleotide that cannot hybridize with the second oligonucleotide or the corresponding nucleic acid base of the target nucleic acid is a mismatched or non-complementary nucleic acid base.

[0050] "Modulating" refers to altering or regulating characteristics in cells, tissues, organs, or organisms. For example, modulating a target nucleic acid may mean increasing or decreasing the level of the target nucleic acid in cells, tissues, organs, or organisms. A "modulator" is a substance that brings about a change in cells, tissues, organs, or organisms. For example, a compound may be a modulator that reduces the amount of a target nucleic acid in cells, tissues, organs, or organisms.

[0051] "MOE" stands for methoxyethyl.

[0052] A "monomer" refers to a single unit of an oligomer. Examples of monomers, though not limited to them, include nucleosides and nucleotides.

[0053] "Motif" refers to the pattern of unmodified and / or modified sugar moieties, nucleic acid bases, and / or nucleoside bonds within an oligonucleotide.

[0054] "Natural" or "natural existence" means something that is found in nature.

[0055] "Non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" refers to a modified sugar moiety that includes modifications such as substituents that do not bridge two atoms of the sugar to form a second ring. "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Examples of nucleic acids, though not limited to them, include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

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

[0057] "Nucleic acid base sequence" refers to the sequence of consecutive nucleic acid bases in a nucleic acid or oligonucleotide, independent of any sugar or nucleoside bonds.

[0058] A "nucleoside" refers to a compound containing a nucleic acid base and a sugar moiety. The nucleic acid base and sugar moiety can be independently either unmodified or modified. A "modified nucleoside" refers to a nucleoside containing a modified nucleic acid base and / or a modified sugar moiety. Modified nucleosides include debasic nucleosides that lack a nucleic acid base.

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

[0060] "Oligonilographic molecule" means a polymer of bonded nucleosides, each of which may be independently modified or unmodified. Unless otherwise specified, oligonucleotides consist of 8 to 80 bonded nucleosides. "Modified oligonucleotide" means an oligonucleotide in which at least one sugar, nucleic acid base, or nucleoside bond has been modified. "Unmodified oligonucleotide" means an oligonucleotide that does not contain any sugar, nucleic acid base, or nucleoside modifications.

[0061] A "parent oligonucleotide" refers to an oligonucleotide whose sequence is similar to that of other oligonucleotides, except for differences in length, motif, and / or chemical structure, and is used as the basis for the design of more oligonucleotides with similar sequences. Newly designed oligonucleotides may have sequences identical to or overlapping with the parent oligonucleotide.

[0062] A "phosphorothioate bond" refers to a modified phosphate bond in which one of the non-bridged oxygen atoms is replaced by a sulfur atom. A phosphorothioate nucleoside bond is a modified nucleoside bond.

[0063] The "phosphorus moiety" refers to an atomic group containing a phosphorus atom. In certain embodiments, the phosphorus moiety includes a monophosphate, diphosphate, or triphosphate, or a phosphorothioate.

[0064] A "part" refers to a specified number of consecutive (i.e., linked) nucleic acid bases. In certain embodiments, a part is a specified number of consecutive nucleic acid bases of a target nucleic acid. In certain embodiments, a part is a specified number of consecutive nucleic acid bases of an oligomeric compound.

[0065] "To reduce" means to make something smaller in range, size, quantity, or number.

[0066] "RNAi compounds" refer to antisense compounds that modulate target nucleic acids and / or proteins encoded by target nucleic acids by acting at least partially via RISC or Ago2, but without RNase H. Examples of RNAi compounds include, but are not limited to, double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, such as microRNA mimetic compounds.

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

[0068] The term "selective" in relation to an effect means that the effect on one cell is greater than that on another cell by any given quantitative range or multiplier. For example, a compound containing a GLP-1 receptor conjugate ligand moiety that is "selective" to cells expressing the GLP-1 receptor, or a compound that "selectively" targets cells expressing the GLP-1 receptor, will target GLP-1 receptor-expressing cells to a greater degree than a compound that does not contain the GLP-1 receptor conjugate ligand moiety. As another example, a compound containing a GLP-1 receptor conjugate ligand moiety that is "selective" to cells expressing the GLP-1 receptor, or a compound that "selectively" targets cells expressing the GLP-1 receptor, will target GLP-1 receptor-expressing cells to a greater degree than cells that do not express the GLP-1 receptor or express it at relatively low levels. It should be understood that the term "selective" does not require absolute all-or-nothing selectivity.

[0069] In relation to a compound, "single-stranded" means that the compound has only one oligonucleotide. "Self-complementary" means that the oligonucleotide hybridizes with itself, at least partially. A compound consisting of one oligonucleotide, where the oligonucleotide 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 nucleic acid base position within a target nucleic acid.

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

[0072] "Specifically inhibiting" a target nucleic acid means reducing or blocking the expression of the target nucleic acid with less or no effect on non-target nucleic acids. Reduction does not necessarily mean complete elimination of the expression of the target nucleic acid.

[0073] "Standard cell assay" refers to the assay described in the examples and its appropriate variations.

[0074] "Standard in vivo experiment" refers to the procedure described in the examples and any reasonable variations thereof.

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

[0076] A "sugar substitute" refers to a modified sugar moiety other than a furanosyl moiety that can bond a nucleic acid base to another group within the oligonucleotide, such as an internucleoside bond, conjugate group, or terminal group. Modified nucleosides containing sugar substitutes can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary compounds or nucleic acids.

[0077] A "target gene" refers to a gene that codes for a target.

[0078] In relation to a target nucleic acid, "targeting" means the specific hybridization of an oligonucleotide to the target nucleic acid that induces the desired effect. In relation to the GLP-1 receptor, "targeting" means the binding of the GLP-1 receptor ligand conjugate to the GLP-1 receptor.

[0079] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to nucleic acids that can be targeted by the compounds described herein.

[0080] The "target region" refers to the portion of a target nucleic acid that one or more compounds target.

[0081] The "target segment" refers to the nucleotide sequence of the target nucleic acid that the compound targets. The "5' target site" refers to the 5' end nucleotide of the target segment. The "3' target site" refers to the 3' end nucleotide of the target segment.

[0082] A "terminal group" refers to a chemical group or atomic group that is covalently bonded to the end 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 binds the oligonucleotide to the GLP-1 receptor ligand conjugate moiety.

[0084] In certain embodiments, the oligonucleotide has a length of 8 to 80 bound nucleosides, 10 to 30 bound nucleosides, 12 to 30 bound nucleosides, or 15 to 30 bound nucleosides.

[0085] In certain embodiments, the oligonucleotide is a modified oligonucleotide comprising at least one modified nucleoside linkage, at least one modified sugar, or at least one modified nucleic acid base. In certain embodiments, the modified nucleoside linkage is a phosphorothioate nucleoside linkage. In certain embodiments, each modified nucleoside linkage in the modified oligonucleotide is a phosphorothioate nucleoside 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 nucleic acid base is 5-methylcytosine.

[0088] In a particular embodiment, the modified oligonucleotide is A gap segment consisting of a bound deoxynucleoside; A 5' wing segment consisting of a bound nucleoside; and 3' wing segment consisting of a bound nucleoside; The gap segment is located directly adjacent to and between the 5' and 3' wing segments, and each nucleoside of each wing segment contains a modified sugar.

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

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

[0091] In certain embodiments, the compound comprises a double helix. In certain embodiments, the double helix comprises a first chain comprising a modified oligonucleotide and a second chain complementary to the first chain. In certain embodiments, the first chain comprising the modified oligonucleotide is complementary to the RNA transcript. In certain embodiments, the second chain is complementary to the RNA transcript. In certain embodiments, the compound comprises a double helix comprising (i) a first chain comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate moiety, and (ii) a second chain complementary to the first chain. In certain embodiments, the compound comprises a double helix comprising (i) a first chain comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate moiety, and (ii) a second chain complementary to the first chain, wherein the first chain is complementary to the RNA transcript. In certain embodiments, the compound comprises a double helix comprising (i) a first chain containing a modified oligonucleotide, optionally a conjugate linker, and a GLP-1 receptor ligand conjugate moiety, and (ii) a second chain complementary to the first chain, the second chain being complementary to an RNA transcript.

[0092] In certain embodiments, the compound is a miRNA mimetic.

[0093] In certain embodiments, the compound comprises a ribonucleotide. In certain embodiments, the compound comprises a deoxyribonucleotide.

[0094] In certain embodiments, the oligonucleotide is complementary to an intracellular RNA transcript, such as that of pancreatic cells or pancreatic β-islet cells.

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

[0096] In certain embodiments, the GLP-1 receptor ligand conjugate moiety is a peptide conjugate moiety targeting the GLP-1 receptor, a small molecule conjugate moiety, an aptamer conjugate moiety, or an antibody conjugate moiety.

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

[0098] In certain embodiments, the GLP-1 peptide conjugate moiety includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid moieties 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 isolength portion of any of the amino acid sequences of SEQ ID NOs. 1 to 57.

[0099] In certain embodiments, the GLP-1 peptide conjugate moiety includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid moieties which are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the isolength portion of any of the amino acid sequences of SEQ ID NOs. 1 to 57.

[0100] In certain embodiments, the GLP-1 peptide conjugate portion is 8 to 50 amino acids long and is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to any of the amino acid sequences of SEQ ID NOs: 1 to 57 over its entire length.

[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 across its entire length to any of the amino acid sequences of SEQ ID NOs: 1 to 57.

[0102] In certain embodiments, the GLP-1 peptide conjugate moiety is GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which in 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 ID 1)) It includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid segments that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the isolength portion of the amino acid sequence of (ru).

[0103] In certain embodiments, the GLP-1 peptide conjugate moiety includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid moieties that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the isolength 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 long and is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1) over its entire length.

[0105] In certain embodiments, the GLP-1 peptide conjugate moiety includes 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 to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1) over its entire length.

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

[0108] In a particular embodiment, 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 contains the amino acid sequence of GLP-1(7-36)amide:HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (which in 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 GLP-1(7-36)amide: (which, in 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 GLP-1(7-36):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (which, in 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 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 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 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)).

[0115] In certain embodiments, the GLP-1 peptide conjugate moiety consists 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)).

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

[0117] In a particular embodiment, the GLP-1 peptide conjugate portion consists of any of the amino acid sequences of SEQ ID NOs: 1 to 57.

[0118] In certain embodiments, the GLP-1 peptide conjugate moiety may be any of the C-terminal amides or acids of SEQ ID NOs: 1 to 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), where Aib is aminoisobutyric acid.

[0120] In a particular embodiment, 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), where 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), where 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), where Aib is aminoisobutyric acid and Pen is penicillamine.

[0123] In certain embodiments, the GLP-1 peptide conjugate moiety can bind to the GLP-1 receptor.

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

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

[0126] In certain embodiments, the cells are located within 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 binds the GLP-1 receptor ligand conjugate moiety to the 5' end of the oligonucleotide.

[0129] In certain embodiments, the conjugate linker binds 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 includes a disulfide bond.

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

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

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

[0135] In a particular embodiment, the conjugate linker comprises three linker-nucleosides.

[0136] In a particular embodiment, the three linker-nucleosides have a TCA motif.

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

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

[0139] In certain embodiments, the conjugate linker contains polyethylene glycol groups.

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

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

[0142] In a particular embodiment, the conjugate linker is [ka] (In the formula, X is attached directly or indirectly to the GLP-1 receptor ligand conjugate portion; and Y attaches directly or indirectly to the modified oligonucleotide. It includes. In certain embodiments, X includes O. In certain embodiments, Y includes a phosphate group. In certain embodiments, X is attached to the GLP-1 receptor ligand conjugate moiety by a disulfide bond.

[0143] In a particular embodiment, the conjugate linker is [ka] (In the formula, X is attached directly or indirectly to the GLP-1 receptor ligand conjugate portion; and T1 contains a modified oligonucleotide; and Bx is a modified or unmodified nucleic acid base. This includes. In certain embodiments, X includes a disulfide bond.

[0144] In a particular embodiment, 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 expressed by the formula: [ka] It is a pyrrolidinyl group having; j is either 0 or 1; n is approximately 1 to approximately 10; p is between 1 and approximately 10; m is 0 or 1-4; and If Y is an amino acid, then m is 1. Includes.

[0145] In certain embodiments, Y is an amino(-NH-) or 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 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 a particular embodiment, the conjugate linker is [ka] Includes.

[0148] In a particular embodiment, the conjugate linker is [ka] Includes.

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

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

[0151] In certain embodiments, the compound comprising the conjugate linker is [ka] (In the ceremony NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the rest of the GLP-1 receptor ligand conjugate moiety; and Y attaches to oligonucleotides directly or indirectly. 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 the expression of a target nucleic acid within a cell includes contacting the cell with a compound of any of the embodiments described above, thereby modulating the expression of the nucleic acid target within the cell. In certain embodiments, the cell expresses a GLP-1 receptor on its surface. In certain embodiments, the cell is a pancreatic cell, such as a β-islet cell. In certain embodiments, the cell is a pituitary cell, a leptomeningeal cell, a central nervous system (CNS) cell, a gastric cell, an intestinal cell, a duodenal cell, an ileal cell, a colon cell, a mammary 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 neurological cancer, including, but not limited to, meningioma, astrocytoma, glioblastoma, ependymoma, and schwannoma. In certain embodiments, cancer is an embryonic carcinoma, including, but not limited to, medulloblastoma, nephroblastoma, and neuroblastoma. In certain embodiments, cancer is, but 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 cells with any of the compounds of the embodiments described above inhibits the expression of a nucleic acid target. In certain embodiments, the nucleic acid target is premRNA, mRNA, non-coding RNA, or miRNA. In certain embodiments, the cells are located within an animal.

[0154] In certain embodiments, a method for modulating the expression of a target nucleic acid in an animal includes modulating the expression of the target nucleic acid in an animal by administering a compound of any of the embodiments described above to the animal. In certain embodiments, the expression of the nucleic acid target is modulated in animal cells expressing a GLP-1 receptor on the cell surface. In certain embodiments, the expression of the nucleic acid target is modulated in pancreatic cells, such as β-islet cells, of an animal. In certain embodiments, the cells are pancreatic cells, such as β-islet cells. In certain embodiments, the cells are pituitary cells, piala cells, duodenal cells, ileal cells, colon cells, mammary cells, lung cells, or kidney cells. In certain embodiments, the cells expressing a GLP-1 receptor on the surface are cancer cells. 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, cancer is a neurological cancer, including, but not limited to, meningioma, astrocytoma, glioblastoma, ependymoma, and schwannoma. In certain embodiments, cancer is an embryonic cancer, including, but not limited to, medulloblastoma, nephroblastoma, and neuroblastoma. In certain embodiments, cancer is a cancer, including, but 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, administration of the compound inhibits the expression of a nucleic acid target in an animal. In certain embodiments, the nucleic acid target is premRNA, mRNA, non-coding RNA, or miRNA.

[0155] This specification also provides the use of the compounds described herein for the manufacture of pharmaceuticals in the treatment of cancer.

[0156] In a particular embodiment, the method for preparing the compound is: [ka] (In the formula, X1 is an oligonucleotide, and the compound is a GLP-1 peptide conjugated oligonucleotide.) This involves reacting it with the GLP-1 peptide.

[0157] In a particular embodiment, the method for preparing the compound is: An oligonucleotide comprising a hexamethyl linker and a terminal amine at the 5' end of the oligonucleotide, is given by formula: [ka] It is reacted with 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) having the formula: [ka] (In the formula, X1 is an oligonucleotide); To obtain compound 2 having, Compound 2 is reacted with the GLP-1 peptide, thereby producing the formula: [ka] (In the formula, X1 is an oligonucleotide, and X2 is a GLP-1 peptide.) To obtain a GLP-1 peptide conjugate oligonucleotide having Includes.

[0158] In a particular embodiment, a method for preparing GLP-1 peptide-conjugated oligonucleotides is: A solution comprising an oligonucleotide having a hexamethyl linker and a terminal amine at the 5' end of the oligonucleotide, is given by formula: [ka] Mix with a solution containing 3-(2-pyridyldithiopropionic acid N-hydroxysuccinimide ester) having the formula: [ka] (In the formula, X1 is an oligonucleotide.) To obtain compound 2 having, A solution containing compound 2 is mixed with a solution containing GLP-1 peptide, thereby producing the formula: [ka] (In the formula, X1 is an oligonucleotide, and X2 is a GLP-1 peptide.) To obtain a GLP-1 peptide conjugate 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 contains acetonitrile and NaHCO3 and has a pH of about 8.0.

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

[0163] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may include at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the isolength portion of any of the amino acid sequences of SEQ ID NOs. 1 to 57.

[0164] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may include at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the isolength portion of any of the amino acid sequences of SEQ ID NOs. 1 to 57.

[0165] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may be 8 to 50 amino acids long and 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% homologous to any of the amino acid sequences of SEQ ID NOs: 1 to 57 over its entire length.

[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 to 57.

[0167] In any of the above methods for preparing a compound or a GLP-1 peptide conjugate oligonucleotide, the GLP-1 peptide is GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which, in 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 It may include at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid segments that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the isolength portion of the amino acid sequence of u-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).

[0168] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may include at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the isolength 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 may be 8 to 50 amino acids long and 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% homologous to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 1) over its entire length.

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

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

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

[0173] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may contain the amino acid sequence of GLP-1(7-36)amide:HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (which, in 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 above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may consist of the amino acid sequence of GLP-1(7-36)amide (SEQ ID NO: 2).

[0175] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may contain the amino acid sequence GLP-1(7-36):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (which in 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 may include 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)).

[0177] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may consist of the amino acid sequence: 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)).

[0178] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may include 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 above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may 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 above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may contain any of the amino acid sequences of SEQ ID NOs: 1 to 57.

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

[0182] In any of the above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may contain 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), where Aib is aminoisobutyric acid.

[0183] In any of the above methods for 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), where Aib is aminoisobutyric acid.

[0184] In any of the aforementioned methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may contain 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 above methods for 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 above methods for preparing a compound or a GLP-1 peptide-conjugated oligonucleotide, the GLP-1 peptide may contain a reactive sulfur moiety.

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

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

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

[0190] In certain embodiments, the compounds described herein include or consist of modified oligonucleotides. In certain embodiments, the modified oligonucleotides have a nucleic acid base sequence complementary to the nucleic acid base sequence of the target nucleic acid.

[0191] In certain embodiments, the compound or antisense compound is single-stranded. Such a single-stranded compound or antisense compound comprises or consists of an oligomeric compound. In certain embodiments, such an oligomeric compound comprises or consists 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 nucleic acid base sequence.

[0192] In certain embodiments, the compound is double-stranded. Such a double-stranded compound comprises a first modified oligonucleotide having a region complementary to the 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, the thymine nucleic acid bases in the modified oligonucleotide are replaced with uracil nucleic acid bases. In certain embodiments, the compound contains 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 bound nucleosides long, and the second modified oligonucleotide is 12 to 30 bound nucleosides long. In certain embodiments, the antisense compound is double-stranded. Such a double-stranded antisense compound comprises a first oligomeric compound having a region complementary to the target nucleic acid, and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric compound of such a double-stranded antisense compound generally contains or consists of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such a double-stranded antisense compound may be modified or unmodified. Either or both of the oligomeric compounds of the double-stranded antisense compound may contain a conjugate group. The oligomeric compounds of the double-stranded antisense compound may contain a non-complementary overhanging nucleoside.

[0193] In certain embodiments, the compound comprises a double helix comprising (i) a first chain comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate moiety, and (ii) a second chain complementary to the first chain. In certain embodiments, the compound comprises a double helix comprising (i) a first chain comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate moiety, and (ii) a second chain complementary to the first chain, wherein the first chain is complementary to the RNA transcript. In certain embodiments, the compound comprises a double helix comprising (i) a first chain comprising a modified oligonucleotide, optionally a conjugate linker and a GLP-1 receptor ligand conjugate moiety, and (ii) a second chain complementary to the first chain, wherein the second chain is complementary to the RNA transcript.

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

[0195] In certain embodiments, the compounds described herein have a nucleic acid base sequence that, when described in the 5' to 3' direction, includes the reverse complementary sequence of the target segment of the target nucleic acid being targeted.

[0196] In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 10 to 30 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 12 to 30 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 12 to 22 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 14 to 30 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 14 to 20 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 15 to 30 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 15 to 20 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 16 to 30 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 16 to 20 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 17 to 30 bonds. In certain embodiments, the compounds described herein include oligonucleotides with a subunit length of 17 to 20 bonds. In certain embodiments, the compounds described herein include oligonucleotides having a subunit length of 18 to 30 units. In certain embodiments, the compounds described herein include oligonucleotides having a subunit length of 18 to 21 units. In certain embodiments, the compounds described herein include oligonucleotides having a subunit length of 18 to 20 units. In certain embodiments, the compounds described herein include oligonucleotides having a subunit length of 20 to 30 units.In other words, such oligonucleotides have lengths of 12-30 bonded subunits, 14-30 bonded subunits, 14-20 subunits, 15-30 subunits, 15-20 subunits, 16-30 subunits, 16-20 subunits, 17-30 subunits, 17-20 subunits, 18-30 subunits, 18-20 subunits, 18-21 subunits, 20-30 subunits, or 12-22 bonded subunits, respectively. In certain embodiments, the compounds described herein include oligonucleotides with a length of 14 bonded subunits. In certain embodiments, the compounds described herein include oligonucleotides with a length of 16 bonded subunits. In certain embodiments, the compounds described herein include oligonucleotides with a length of 17 bonded subunits. In certain embodiments, the compounds described herein include oligonucleotides with a length of 18 bonded subunits. In certain embodiments, the compounds described herein include oligonucleotides with a length of 19 bonded subunits. In certain embodiments, the compounds described herein include oligonucleotides with a length of 20 bonded subunits. In another embodiment, the compounds described herein include oligonucleotides with 8-80, 12-50, 13-30, 13-50, 14-30, 14-50, 15-30, 15-50, 16-30, 16-50, 17-30, 17-50, 18-22, 18-24, 18-30, 18-50, 19-22, 19-30, 19-50, or 20-30 binding subunits. In certain such embodiments, the compounds described herein include oligonucleotides having binding subunit lengths in the range defined by 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 any two of the above values.In some embodiments, the binding subunit is a nucleotide, nucleoside, or nucleic acid base.

[0197] In certain embodiments, the compound may further include additional features or elements, such as a conjugate group attached to the oligonucleotide. In certain embodiments, such a compound is an antisense compound. In certain embodiments, such a compound is an oligomeric compound. In embodiments in which the conjugate group includes a nucleoside (i.e., a nucleoside that binds 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, compounds 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). Shortened or truncated compounds targeting nucleic acids may have two subunits deleted from the 5' end of the compound, or alternatively, two subunits deleted from the 3' end. Alternatively, the deleted nucleoside may be dispersed throughout the compound.

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

[0200] It is possible to increase or decrease the length of compounds such as oligonucleotides and / or introduce mismatched bases without eliminating their 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 motifs can result in one or more significant differences in 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 partially via the RISC pathway to degrade and / or blockade target nucleic acids (hence, microRNA / microRNA mimetic compounds). As used herein, the term siRNA is 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, post-transcriptional gene silencing RNA (ptgsRNA), etc. Furthermore, as used herein, the term "RNAi" is equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translation inhibition, or epigenetics.

[0202] In certain embodiments, the first strand of the compound is a siRNA guide strand and the second strand of the compound is a siRNA passenger strand. In certain embodiments, the second strand of the compound is complementary to the first strand. In certain embodiments, 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 include 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 forms, the stereoisomeric forms of which can be defined as (R) or (S) from the perspective of absolute stereochemistry, e.g., α or β with respect to sugar anomers or (D) or (L) with respect to amino acids, etc. The modified oligonucleotides provided herein include all such possible isomers in their racemic and optically pure forms, unless otherwise specified. Similarly, all cis- and trans-isomers as well as tautomers are 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 element. For example, compounds herein that include hydrogen atoms include all possible deuterium substitutions of each 1 H hydrogen atom. Isotope substitutions included by the compounds herein include, but are not limited to, 1 in place of 2 H or 3 H, 12 in place of 13 C or 14 C, 14 in place of 15 N, 16 in place of 17 O or 18 O and 32 in place of 33 S, 34 S, 35 S or 36S is one example. In certain embodiments, non-radioactive isotope substitution can impart new properties to compounds that are beneficial for use as therapeutic or research tools. In certain embodiments, radioactive isotope substitution can produce compounds suitable for research or diagnostic purposes, such as imaging assays.

[0205] Specific mechanism In certain embodiments, the compounds described herein include or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds include oligomeric compounds. In certain embodiments, the compounds described herein are capable of hybridizing to a target nucleic acid to yield at least one antisense activity. In certain embodiments, the compounds described herein selectively affect one or more target nucleic acids. Such compounds include nucleic acid sequences that hybridize to one or more target nucleic acids to yield one or more desired antisense activities, and do not hybridize to one or more non-target nucleic acids, or do not hybridize to one or more non-target nucleic acids in a manner that yields undesirable significant antisense activity.

[0206] In certain antisense activities, hybridization of the compounds described herein to target nucleic acids results in the recruitment of proteins that cleave 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 double helix. The DNA in such an RNA:DNA double helix 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 gapmer gap are tolerable.

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

[0208] In certain embodiments, hybridization of the compounds described herein to a target nucleic acid does not result in the recruitment of proteins that cleave the target nucleic acid. In certain such embodiments, hybridization of the compounds to a target nucleic acid results in a change in the splicing of the target nucleic acid. In certain embodiments, hybridization of the compounds to a target nucleic acid results in the inhibition of binding interactions between the target nucleic acid and proteins or other nucleic acids. In certain such embodiments, hybridization of the compounds to a target nucleic acid results in a change in the translation of the target nucleic acid.

[0209] Antisense activity can be observed directly or indirectly. In certain embodiments, observation or detection of antisense activity includes observing or detecting changes in the amount of target nucleic acid or protein encoded by such target nucleic acid, changes in the ratio of splice variants of nucleic acid or protein, and / or phenotypic changes in cells or animals.

[0210] Target nucleic acid, target region, and nucleotide sequence 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 comprising introns, exons, and untranslated regions. In certain embodiments, the target RNA is mRNA. In certain embodiments, the target nucleic acid is pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron / exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is present within a cell expressing the GLP-1 receptor. In certain embodiments, the GLP-1 receptor-expressing cells are pancreatic cells such as β-islet cells.

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

[0212] Hybridization can occur under variable conditions. Hybridization conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules being 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 described as complementary to another nucleic acid if the nucleic acid base sequence of such oligonucleotide or one or more regions thereof matches the nucleic acid base sequence of another oligonucleotide or nucleic acid or one or more regions thereof when these two nucleic acid base sequences are aligned in opposite directions. Unless otherwise specified, the nucleic acid base matches or complementary nucleic acid bases 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). Complementary oligonucleotides and / or nucleic acids do not need to have nucleic acid base complementarity at each nucleoside and may contain one or more nucleic acid base mismatches. An oligonucleotide is fully complementary or 100% complementary if such oligonucleotide has nucleic acid base matches at each nucleoside without any nucleic acid base mismatches.

[0215] In certain embodiments, the compounds described herein include or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds include oligomeric compounds. Non-complementary nucleic acid bases between the compound and the target nucleic acid may be tolerable if they remain in a state where they can specifically hybridize to the target nucleic acid. Furthermore, since the compound can hybridize across one or more segments of the target nucleic acid, intervening or adjacent segments do not participate in the hybridization event (e.g., loop structures, mismatches, or hairpin structures).

[0216] In certain embodiments, the compounds or specific portions thereof provided herein are complementary to the target nucleic acid, target region, target segment, or specific portion thereof by at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or up to these percentages. In certain embodiments, the compounds or specific portions thereof provided herein are complementary to the target nucleic acid, target region, target segment, or specific portion thereof by 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, or any number within these ranges. The percentage of complementarity of the compound with the target nucleic acid can be determined using common methods.

[0217] For example, a compound in which 18 of its 20 nucleic acid bases are complementary to the target region and therefore specifically hybridizes will exhibit 90 percent complementarity. In this example, the remaining non-complementary nucleic acid bases can cluster or be scattered among the complementary nucleic acid bases and do not need to be consecutive with each other or with complementary nucleic acid bases. Thus, a compound with an 18-nucleotide length having four non-complementary nucleic acid bases adjacent to two regions that are perfectly complementary to the target nucleic acid will have 77.8% overall complementarity with the target nucleic acid. The percentage complementarity of a compound with a region of the target nucleic acid can be systematically determined using the 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), which are known in the art. Homology percentages, sequence identity percentages, or complementarity percentages 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 employs the Smith and Waterman algorithm (Adv.Appl.Math., 1981, 2, 482 489).

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

[0219] In certain embodiments, the compounds described herein contain one or more nucleic acid bases that are mismatched with respect to a target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against non-targets is reduced more significantly. Thus, in certain such embodiments, the selectivity of the compound is improved. In certain embodiments, the mismatch is specifically located within oligonucleotides having a gapmer motif. In certain such embodiments, the mismatch is 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 mismatch is 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 mismatch is located at positions 1, 2, 3, or 4 from the 5' end of the wing region. In certain such embodiments, the mismatch is located at positions 4, 3, 2, or 1 from the 3' end of the wing region. In certain embodiments, the mismatch is specifically located within oligonucleotides that do not have a gapmer motif. In certain such embodiments, the mismatch is located 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 located at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3' end of the oligonucleotide.

[0220] The localization of non-complementary nucleic acid bases may be at the 5' or 3' end of the compound. Alternatively, one or more non-complementary nucleic acid bases may be located internally within the compound. If two or more non-complementary nucleic acid bases are present, they may be contiguous (i.e., bonded) or discontinuous. In one embodiment, the non-complementary nucleic acid bases are localized within the wing segment of a gapmer oligonucleotide.

[0221] In certain embodiments, the compounds described herein, having a nucleic acid base length of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a maximum of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, contain four or fewer, three or fewer, two or fewer, or one or fewer non-complementary nucleic acid bases with respect to the target nucleic acid, e.g., the target nucleic acid or a defined portion thereof.

[0222] In certain embodiments, the compounds described herein, having a nucleic acid base length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid base lengths, or up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid base lengths, contain six or fewer, five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer non-complementary nucleic acid bases with respect to the target nucleic acid, e.g., the target nucleic acid or a defined portion thereof.

[0223] In certain embodiments, the compounds described herein may be complementary to a portion of the target nucleic acid. As used herein, “a portion” refers to a defined number of consecutive (i.e., bound) nucleic acid bases within a region or segment of the target nucleic acid. “A portion” may also refer to a defined number of consecutive nucleic acid bases of a compound. In certain embodiments, the compound is complementary to a portion of at least eight nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least nine nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least ten nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least eleven nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least twelve nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least thirteen nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least fourteen nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a portion of at least fifteen nucleic acid bases of the target segment. In certain embodiments, the compound is complementary to a subset of at least 16 nucleic acid bases of the target segment. Compounds complementary to a subset of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid bases of the target segment, or to any two of their values, are also intended.

[0224] identity The compounds provided herein may also have a defined percentage identity with respect to a compound, or part thereof, represented by a specific nucleotide sequence, sequence number, 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. Compounds used herein are identical to the sequences disclosed herein if they have the same nucleic acid base pairing ability. For example, an RNA containing uracil instead of thymidine in the disclosed DNA sequence would be considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shorter and longer forms of the compounds described herein, as well as compounds having non-identical bases to the compounds provided herein, are also conceivable. Non-identical bases may be adjacent to each other or distributed throughout the compound. The percentage identity of a compound is calculated according to the number of bases that have the same base pairs as the sequence it is being compared to.

[0225] In certain embodiments, a compound or portion thereof described herein is identical to at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or up to one or more of the compounds or sequence numbers or portions thereof disclosed herein. In certain embodiments, a compound described herein is identical to a compound or portion thereof represented by a specific nucleotide sequence, sequence number, or specific ISIS or ION number by about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any percentage between those values, and the compound comprises an oligonucleotide having one or more mismatched nucleic acid bases. In certain such embodiments, the mismatch is located 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 located 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 include or consist of an antisense compound. In certain embodiments, a portion of the antisense compound is compared to an isolength portion of the target nucleic acid. In certain embodiments, the 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleic acid base portions are compared to an isolength portion of the target nucleic acid.

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

[0228] Specific modified compounds In certain embodiments, the compounds described herein include or consist of an oligonucleotide comprising a bound nucleoside. The oligonucleotide may be an unmodified oligonucleotide (RNA or DNA) or a modified oligonucleotide. The modified oligonucleotide comprises at least one modification to the unmodified RNA or DNA (i.e., at least one modified nucleoside (including a modified sugar moiety and / or a modified nucleic acid base) and / or at least one modified nucleoside bond).

[0229] A. Modified nucleoside Modified nucleosides include either a modified sugar moiety, a modified nucleic acid base, or both.

[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 substitute. Such a sugar substitute may include one or more substitutions corresponding to substitutions of other types of modified sugar moieties.

[0231] In certain embodiments, the modified sugar moiety comprises a bicyclic modified sugar moiety containing a furanosyl ring having one or more acyclic substituents, the 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 bicyclic modified sugar moiety are branched. Examples of suitable 2'- substituents for the bicyclic modified sugar moiety include, but not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2'- substituents are halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O-C1~C 10 Alkoxy, O-C1~C 10 Substitutive alkoxy, O-C1~C 10 Alkyl, O-C1~C 10 Substitutive alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-Alkenyl, O-Alkinyl, S-Alkinyl, N(R m )-Alkynyl, O-Alkyrenyl-O-alkyl, Alkynyl, Alkalyl, Aralkyl, O-Alkalyl, O-Aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ) or OCH2C(=O)-N(R m )(R n ) are selected from the above, and in the formula, each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C1. 10These are alkyl and 2'-substituents described in Cook et al.'s U.S. Patent No. 6,531,584; Cook et al.'s U.S. Patent No. 5,859,221; and Cook et al.'s U.S. Patent No. 6,005,087. Specific embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Suitable 4'-substituents for the linear non-bicyclic modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al.'s International Publication No. 2015 / 106128. Suitable 5'-substituents for the non-bicyclic modified sugar moiety include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugar comprises two or more non-crosslinked sugar substituents, such as a 2'-F-5'-methyl sugar moiety, and modified sugar moieties and modified nucleosides described in International Publication No. 2008 / 101157 by Migawa et al. and U.S. Patent Application Publication No. 2013 / 0203836 by Rajeev et al.

[0232] In certain embodiments, the 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside is 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 )) contains a sugar moiety with a linear 2'- substituent selected from each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C1. 10 It is alkyl.

[0233] In certain embodiments, a 2'-substituted nucleoside or a 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising 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, a 2'-substituted nucleoside or a 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent selected from F, OCH3, and OCH2CH2OCH3.

[0235] Nucleosides comprising a modified sugar moiety, such as a non-bicyclic modified sugar moiety, are named according to the position of the substitution on the sugar moiety of the nucleoside. For example, a nucleoside comprising a 2'-substituted or 2-modified sugar moiety is referred to as a 2'-substituted nucleoside or a 2-modified nucleoside.

[0236] Certain modified sugar moieties include bridging sugar substituents that form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes bridging between 4' and 2' furanose ring atoms. Examples of such 4'-to-2' cross-linked sugar substituents include, but are not limited to, 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "bound ethyl" or "cEt" in the S configuration), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("bound MOE" or "cMOE") and their analogues (e.g., U.S. Patent No. 7,399,845 by Seth et al., U.S. Patent No. 7,569,686 by Bhat et al., Swayze See U.S. Patent No. 7,741,457 and U.S. Patent No. 8,022,193 by Swayze et al.), 4'-C(CH3)(CH3)-O-2' and its analogues (see, for example, U.S. Patent No. 8,278,283 by Seth et al.), 4'-CH2-N(OCH3)-2' and its analogues (see, for example, U.S. Patent No. 8,278,425 by Prakash et al.), 4'-CH2-ON(CH3)-2' (see, for example, U.S. Patent No. 7,696,345 and U.S. Patent No. 8,124,745 by Allerson et al.), 4'-CH2-C(H)(CH3)-2' (see, for example, Zhou, et al. See al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and its analogues (see, for example, U.S. Patent No. 8,278,426 by 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 These are independently H, protecting groups or C1-C 12Examples include alkyl groups (see, for example, U.S. Patent No. 7,427,672 by Imanishi et al.).

[0237] In certain embodiments, such 4' to 2' crosslinking is independently performed by -[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 )- comprises 1 to 4 bonding groups independently selected from, in the formula, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R a and R b These are independently H, protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12 Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution 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 independent of H, C1~C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution 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 group 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.,1998,8,219-222; 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 by Wengel et al., U.S. Patent No. 6,268,490 by Imanishi et al., U.S. Patent No. 6,770,748 by Imanishi et al., U.S. Reissue Patent No. 44,779 by Imanishi et al.; U.S. Patent No. 6,794,499 by Wengel et al., U.S. Patent No. 6,670,461 by Wengel et al. U.S. Patent No. 7,034,133; U.S. Patent No. 8,080,644 by Wengel et al.; U.S. Patent No. 8,034,909 by Wengel et al.; U.S. Patent No. 8,153,365 by Wengel et al.; U.S. Patent No. 7,572,582 by Wengel et al.; and U.S. Patent No. 6,525,191 by Ramasamy et al., International Publication No. 2004 / 106356 by Torsten et al., International Publication No. Pamphlet No. 91999 / 014226; International Publication No. 2007 / 134181 by Seth et al.; U.S. Patent No. 7,547,684 by Seth et al.; U.S. Patent No. 7,666,854 by Seth et al.; U.S. Patent No. 8,088,746 by Seth et al.; U.S. Patent No. 7,750,131 by Seth et al.; U.S. Patent No. 8,030,467 by Seth et al.; U.S. Patent No. 8,268,980 See the following specifications; Seth et al.'s U.S. Patent No. 8,546,556; Seth et al.'s U.S. Patent No. 8,530,640; Migawa et al.'s U.S. Patent No. 9,012,421; Seth et al.'s U.S. Patent No. 8,501,805; and the U.S. Patent Publication No. 2008 / 0039618 by Allerson et al. and U.S. Patent Publication No. 2015 / 0191727 by Migawa et al.

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

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

[0241] In certain embodiments, the modified sugar moiety is a sugar substitute. In certain such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such a modified sugar moiety also includes the crosslinked and / or non-crosslinked substituents described herein. For example, certain sugar substitutes include substitutions of the 4'-sulfur atom and the 2'-position (see, for example, U.S. Patent No. 7,875,733 and U.S. Patent No. 7,939,677 of Bhat et al.) and / or the 5' position.

[0242] In certain embodiments, the sugar substitute comprises a ring having more than five atoms. For example, in certain embodiments, the sugar substitute comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), mannitol nucleic acid ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA: [ka] (See “F-HNA,” for example, U.S. Patent No. 8,088,904 by Swayze et al.; U.S. Patent No. 8,440,803 by Swayze et al.; U.S. Patent No. 9,005,906 by Swayze et al. F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran), and formula: [ka] (In the formula, independently of each of the modified THP nucleosides, Bx is the nucleic acid base portion; T3 and T4 are, independently, internucleoside binding groups that bind a modified THP nucleoside to the remainder of the oligonucleotide, or one of T3 and T4 is an internucleoside binding group that binds a modified THP nucleoside to the remainder of the oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a binding 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 of J1, J2, and J3 is independently H or C1-C6 alkyl. Examples include nucleosides containing additionally modified THP compounds having [specific properties].

[0243] In certain embodiments, modified THP nucleosides are provided in which 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 not 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 in which 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 substitute comprises a ring having six or more atoms and two or more heteroatoms. For example, its use in nucleosides and oligonucleotides containing a morpholino sugar moiety has been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and U.S. Patent Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506 by Summerton et al.). The term "morpholino" as used herein refers to the following structure: [ka] This refers to a sugar substitute having [the specified properties]. In certain embodiments, morpholino can be modified, for example, by adding or altering substituents from the morpholino structure. Such sugar substitutes are referred to herein as "modified morpholino".

[0245] In certain embodiments, the sugar substitute includes an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al.'s International Publication No. 2011 / 133876.

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

[0247] Modified nucleic acid bases Modifications or substitutions of nucleic acid bases (or bases) are structurally distinct from naturally occurring or synthetically unmodified nucleic acid bases, but functionally interchangeable with them. Both naturally occurring and modified nucleic acid bases can participate in hydrogen bonding. Such nucleic acid base modifications can confer nuclease stability, binding affinity, or some other beneficial biological property to antisense compounds.

[0248] In certain embodiments, the compounds described herein include modified oligonucleotides. In certain embodiments, the modified oligonucleotide includes one or more nucleosides containing unmodified nucleic acid bases. In certain embodiments, the modified oligonucleotide includes one or more nucleosides containing modified nucleic acid bases. In certain embodiments, the modified oligonucleotide includes one or more nucleosides that do not contain nucleic acid bases, referred to as debased nucleosides.

[0249] In certain embodiments, the modified nucleic acid bases are 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 nucleic acid bases are 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-azocymine, 5-ribosyluracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thiolalkyl, 8-hydroxy Selected from syl, 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-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases and fluorinated bases. Further modified nucleic acid bases include tricyclic pyrimidines, such as 1,3-diazaphenoxadin-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxadin-2-one (G-clamp). Modified nucleic acid bases can also be those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808 by 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 teaching the preparation of specific and other modified nucleic acid bases include, but are not limited to, those by Manoharan et al., U.S. Patent Application Publication No. 2003 / 0158403, 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, U.S. Patent No. 175,273; U.S. Patent No. 5,367,066 by Urdea et al.; U.S. Patent No. 5,432,272 by Benner et al.; U.S. Patent No. 5,434,257 by Matteucci et al.; U.S. Patent No. 5,457,187 by Gmeiner et al.; U.S. Patent No. 5,459,255 by Cook et al.; U.S. Patent No. 5,484,908 by Froehler et al.; U.S. Patent No. 5,502,177 by Matteucci et al.; U.S. Patent No. 5,525,711 by 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, Examples include U.S. Patent No. 750,692; U.S. Patent No. 5,948,903 by Cook et al.; U.S. Patent No. 5,587,470 by Cook et al.; U.S. Patent No. 5,457,191 by Cook et al.; U.S. Patent No. 5,763,588 by Matteucci et al.; U.S. Patent No. 5,830,653 by Froehler et al.; U.S. Patent No. 5,808,027 by Cook et al.; U.S. Patent No. 6,166,199 by Cook et al.; and U.S. Patent No. 6,005,096 by Matteucci et al.

[0251] In certain embodiments, the compound targeting the target nucleic acid comprises one or more modified nucleic acid bases. In certain embodiments, the modified nucleic acid base is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

[0252] 3. Inter-modified nucleotide bonding The naturally occurring nucleoside bond in RNA and DNA is a 3'-to-5' phosphodiester bond. In certain embodiments, compounds described herein having one or more modified (i.e., unnatural) nucleoside bonds are often preferred over compounds having naturally occurring nucleoside bonds due to desired properties such as improved cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0253] In certain embodiments, the compound targeting the target nucleic acid comprises one or more modified nucleoside bonds. In certain embodiments, the modified nucleoside bonds are phosphorothioate bonds. In certain embodiments, each nucleoside bond in the antisense compound is a phosphorothioate nucleoside bond.

[0254] In certain embodiments, the compounds described herein include oligonucleotides. Oligonucleotides having modified internucleoside bonds include internucleoside bonds that hold phosphorus atoms and internucleoside bonds that do not contain phosphorus atoms. Typical phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.

[0255] In certain embodiments, the nucleosides of modified oligonucleotides may be linked to one another using any internucleoside bond. Two main classes of internucleoside bond groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphates (also called unmodified or naturally occurring bonds), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S") and phosphorodithioates ("HS-P=S") containing phosphodiester bonds ("P=O"). Representative non-phosphorus-containing nucleoside interbonding groups include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to naturally occurring phosphate bonds, modified nucleoside interbonding groups can be used to alter and generally increase the nuclease resistance of oligonucleotides. In certain embodiments, nucleoside interbonding groups having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral nucleoside interbonding groups include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing nucleoside interbonding groups are well known to those skilled in the art.

[0256] Examples of neutral nucleoside interbonding include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Further examples of neutral nucleoside bonds include nonionic bonds containing siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, for example, Carbohydrate Modifications in Antisense Research; YSSanghvi and PDCook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further examples of neutral nucleoside bonds include nonionic bonds containing mixed N, O, S, and CH2 component moieties.

[0257] In certain embodiments, the oligonucleotide includes modified nucleoside bonds arranged along the oligonucleotide or its region in a defined pattern or modified nucleoside bond motif. In certain embodiments, the nucleoside bonds are arranged in a gapped motif. In such embodiments, the nucleoside bonds in each of the two wing regions are different from the nucleoside bonds in the gap region. In certain embodiments, the nucleoside bonds in the wings are phosphodiesters, and the nucleoside bonds in the gap are phosphorothioates. Since the nucleoside motifs are independently selected, such oligonucleotides having a gapped nucleoside bond motif may or may not have the gapped nucleoside motif, and if the gapped nucleoside motif is present, the wing length and the gap length may or may not be the same.

[0258] In certain embodiments, the oligonucleotide includes a region having alternating nucleoside-linking motifs. In certain embodiments, the oligonucleotide includes a region of uniformly modified nucleoside-linking. In certain such embodiments, the oligonucleotide includes a region uniformly linked by phosphorothioate nucleoside-linking. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioates. In certain embodiments, each nucleoside-linking bond of the oligonucleotide is selected from phosphodiesters and phosphorothioates. In certain embodiments, each nucleoside-linking bond of the oligonucleotide is selected from phosphodiesters and phosphorothioates, and at least one nucleoside-linking bond is a phosphorothioate.

[0259] In certain embodiments, the oligonucleotide comprises at least six phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least eight phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least ten phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least six consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least eight consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least ten consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of twelve consecutive phosphorothioate nucleoside bonds. 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 from the 3' end of the oligonucleotide.

[0260] In certain embodiments, the oligonucleotide comprises one or more methyl phosphate bonds. In certain embodiments, the oligonucleotide having a gap manucleoside motif comprises a bonding motif containing all phosphorothioate bonds except for one or two methyl phosphate bonds. In certain embodiments, one methyl phosphate is located in the central gap of the oligonucleotide having a gap manucleoside motif.

[0261] In certain embodiments, it is desirable that the number of phosphorothioate nucleoside bonds and phosphodiester nucleoside bonds be arranged to maintain nuclease resistance. In certain embodiments, it is desirable that the number and position of phosphorothioate nucleoside bonds and the number and position of phosphodiester nucleoside bonds be arranged to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate nucleoside bonds may be reduced and the number of phosphodiester nucleoside bonds may be increased. In certain embodiments, it is desirable that the number of phosphorothioate nucleoside bonds be reduced and the number of phosphodiester nucleoside bonds be increased while still maintaining nuclease resistance. In certain embodiments, it is desirable that the number of phosphorothioate nucleoside bonds be reduced while maintaining nuclease resistance. In certain embodiments, it is desirable that the number of phosphodiester nucleoside bonds be increased while maintaining nuclease activity.

[0262] 4. Specific motifs In certain embodiments, the compounds described herein include oligonucleotides. Oligonucleotides may have motifs, such as unmodified and / or modified sugar moieties, nucleic acid bases, and / or nucleoside bond patterns. In certain embodiments, a modified oligonucleotide comprises one or more modified nucleosides containing modified sugars. In certain embodiments, a modified oligonucleotide comprises one or more modified nucleosides containing modified nucleic acid bases. In certain embodiments, a modified oligonucleotide comprises one or more modified nucleoside bonds. In such embodiments, the modifications, unmodified and differentially modified sugar moieties, nucleic acid bases, and / or nucleoside bonds of the modified oligonucleotide define the pattern or motif. In certain embodiments, the sugar moieties, nucleic acid bases, and nucleoside bond patterns are independent of each other. Thus, a modified oligonucleotide can be described by its sugar motif, nucleic acid base motif, and / or nucleoside bond motif (as used herein, the nucleic acid base motif describes a modification of the nucleic acid base independent of the sequence of the nucleic acid base).

[0263] 1. Specific sugar motifs In certain embodiments, the compounds described herein include oligonucleotides. In certain embodiments, the oligonucleotide includes one or more types of modified sugars and / or unmodified sugar moieties arranged along the oligonucleotide or its region in a defined pattern or sugar motif. In certain examples, such sugar motifs include, but are not limited to, any of the sugar modifications considered herein.

[0264] In certain embodiments, the modified oligonucleotide comprises or consists of a region having a gapmer motif comprising two external regions or "wings" and a central or internal region or "gap". The three regions of the gapmer motif (5'-wing, gap, and 3'-wing) form a continuous sequence of nucleosides, and at least a portion of the sugar moieties of each nucleoside in the wings differs from at least a portion of the sugar moieties of the nucleosides in the gap. Specifically, at least the sugar moieties of the nucleosides in each wing closest to the gap (the 3'-side nucleoside of the 5'-wing and the 5'-side nucleoside of the 3'-wing) differ from the sugar moieties of the adjacent gap nucleosides, thus defining a boundary between the wings and the gap (i.e., a wing / gap junction). In certain embodiments, the sugar moieties within the gap are identical to each other. In certain embodiments, the gap comprises one or more nucleosides having sugar moieties that differ 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 gapmer). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmer).

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

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

[0267] In certain embodiments, the gapmer is a deoxygapmer. In such embodiments, the nucleoside on the gap side of each wing / gap junction is an unmodified 2'-deoxynucleoside, and the nucleoside on the wing side of each wing / gap junction is a modified nucleoside. In 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, the modified oligonucleotide has a fully modified sugar motif, and each nucleoside of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, the modified oligonucleotide includes or comprises a region having a fully modified sugar motif, and each nucleoside of the region contains a modified sugar moiety. In certain embodiments, the modified oligonucleotide includes or comprises a region having a fully modified sugar motif, and each nucleoside within the fully modified region contains the same modified sugar moiety, which is referred to herein as a homogeneous modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a homogeneous modified oligonucleotide. In certain embodiments, each nucleoside of the homogeneous modification contains the same 2'-modification.

[0269] 2. Specific nucleic acid base motifs In certain embodiments, the compounds described herein include oligonucleotides. In certain embodiments, the oligonucleotide includes modified and / or unmodified nucleic acid bases arranged along the oligonucleotide or its region in a defined pattern or motif. In certain embodiments, each nucleic acid base is modified. In certain embodiments, none of the nucleic acid bases are modified. In certain embodiments, each purine or 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 nucleic acid bases in the modified oligonucleotide are 5-methylcytosine.

[0270] In certain embodiments, the modified oligonucleotide includes a block of modified nucleic acid bases. In certain such embodiments, the block is located at the 3' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleosides of the 3' end of the oligonucleotide. In certain embodiments, the block is located at the 5' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleosides of the 5' end of the oligonucleotide.

[0271] In certain embodiments, the oligonucleotide having a gapmer motif comprises a nucleoside containing a modified nucleic acid base. In certain such embodiments, one nucleoside containing a modified nucleic acid base is located 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 nucleic acid base is selected from 2-thiopyrimidine and 5-propympyrimidine.

[0272] 3. Specific nucleoside bond motifs In certain embodiments, the compounds described herein include oligonucleotides. In certain embodiments, the oligonucleotide includes modified and / or unmodified nucleoside bonds arranged along the oligonucleotide or its region in a defined pattern or motif. In certain embodiments, essentially each nucleoside bond is a phosphate nucleoside bond (P=O). In certain embodiments, each nucleoside bond of the modified oligonucleotide is a phosphorothioate (P=S). In certain embodiments, each nucleoside bond of the modified oligonucleotide is independently selected from phosphorothioate and phosphate nucleoside bonds. In certain embodiments, the sugar motif of the modified oligonucleotide is a gapmer, and all nucleoside bonds within the gap are modified. In certain such embodiments, some or all of the nucleoside bonds in the wing are unmodified phosphate bonds. In certain embodiments, the terminal nucleoside bonds are modified.

[0273] 5. Specific modified oligonucleotides In certain embodiments, the compounds described herein include modified oligonucleotides. In certain embodiments, the above modifications (sugars, nucleic acid bases, nucleoside bonds) are incorporated into the modified oligonucleotide. In certain embodiments, the modified oligonucleotide is characterized by its modifications, motif, and full length. In certain embodiments, such parameters are independent of each other. Thus, unless otherwise indicated, each nucleoside bond of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modification. For example, nucleoside bonds within the wing region of a sugar gapmer may be identical or different from those within the gap region of the sugar motif. Similarly, such a gapmer oligonucleotide may contain one or more modified nucleic acid bases independent of the gapmer pattern of the sugar modification. Furthermore, in certain examples, the oligonucleotide is described by its full length or range and by the length or length range of two or more regions (e.g., regions of nucleosides having a particular sugar modification), in which case it may be possible to select a number of ranges that result in oligonucleotides having full length not included in a particular range. In such cases, both elements must be satisfied. For example, in a particular embodiment, the modified oligonucleotide consists of 15 to 20 bound nucleosides and has a sugar motif consisting of three regions A, B, and C, where region A consists of 2 to 6 bound nucleosides having a specific sugar motif, region B consists of 6 to 10 bound nucleosides having a specific sugar motif, and region C consists of 2 to 6 bound nucleosides having a specific sugar motif. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 bound nucleosides and B consists of 10 bound 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 is 22, which exceeds the upper limit (20) of the total length of modified oligonucleotides.In this specification, if an oligonucleotide is described with respect to one or more parameters, those parameters are not limited. Therefore, a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any length, nucleoside-binding motif, and nucleic acid base motif. Unless otherwise indicated, all modifications are independent of the nucleic acid base sequence.

[0274] Specific 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 comprises one or more conjugate moieties and a conjugate linker that binds the conjugate moieties to an oligonucleotide. The conjugate group may be attached to either or both ends and / or any internal position of the oligonucleotide. In certain embodiments, the conjugate group is attached to the 2' position of the nucleoside of the modified oligonucleotide. In certain embodiments, a conjugate group attached to either or both ends of the oligonucleotide is a terminal group. In certain such embodiments, the conjugate group or terminal group is attached to the 3' and / or 5' ends of the oligonucleotide. In certain such embodiments, the conjugate group (or terminal group) is attached to the 3' end of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 3' end of the oligonucleotide. In certain embodiments, the conjugate group (or terminal group) is attached to the 5' end 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, conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more independently modified or unmodified nucleosides.

[0276] GLP-1 receptor ligand conjugate portion 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 binds 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 portion 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 and 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 can bind to the GLP-1 receptor can be used in some embodiments. For example, in certain embodiments, the small molecule conjugate portion capable of binding to the GLP-1 receptor is described in Willard et al., "Small Molecule Drug Discovery at the Glucagon-like Peptide-1 Receptor," Experimental Diabetes Research Vol.2012 pgs.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 pgs.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 These are small molecule GLP-1 receptor antagonists described in Pharmacologica Sinica (2010) 31:1026-1030 (these are incorporated herein by reference as a whole).

[0279] In a particular embodiment, the small molecule conjugate moiety capable of binding to the GLP-1 receptor is given by the following formula: [ka] [ka] [ka] It ends with either of the following.

[0280] 2. Specific GLP-1 receptor antibody conjugate portion 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 capable of binding to the GLP-1 receptor known in the art 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 as described in International Publication No. 2005018536, U.S. Patent Application Publication No. 20060275288, U.S. Patent No. 8,389,689, or International Publication No. 2011056644 (these are incorporated herein by reference as a whole). In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and an antibody or fragment thereof capable of binding to a GLP-1 receptor as described in International Publication No. 2005018536, U.S. Patent Application Publication No. 20060275288, U.S. Patent No. 8,389,689, or International Publication No. 2011056644 (these are incorporated herein by reference as a whole).

[0281] 3. Specific GLP-1 peptide conjugate moiety 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 may 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 International Publication No. 2007 / 124461 (these are incorporated herein by reference as a whole). 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 International Publication No. 2007 / 124461 (these are incorporated herein by reference as a whole).

[0282] In certain embodiments, the compound is oligonucleotide and GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which, in 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) It comprises a GLP-1 peptide conjugate moiety containing at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive amino acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the isolength portion of the amino acid sequence of (number 1). 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 acid moieties that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the isolength portion of the amino acid sequence of GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (SEQ ID NO: 1), where NH2 represents a C-terminal amide.

[0283] In certain embodiments, the compound is an oligonucleotide, a conjugate linker, and GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (which, in 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- It contains 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 acid portions that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the isolength 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 acid moieties that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to the isolength portion of the amino acid sequence of GLP-1(7-37):HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (SEQ ID NO: 1), where NH2 represents a C-terminal amide.

[0284] In certain embodiments, the compound includes 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 acid moieties that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotide and the isolength 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 acid moieties that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the isolength 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 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 the amino acid sequence of GLP-1(7-37)(SEQ ID NO: 1) over its entire length.

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

[0287] In certain embodiments, the compound comprises an oligonucleotide and a GLP-1 peptide conjugate moiety containing 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).

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

[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 a 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 a 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 a 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 a 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 a 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 a 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 a 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 a C-terminal amide.

[0295] In certain embodiments, the compound may be an oligonucleotide, optionally a conjugate linker, and, but is not limited to, liraglutide (VICTOZA® of Novo Nordisk); albiglutide (SYNCRIA® of 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(7-38);Arg26,34,Lys36(Nε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-36);Aib8.35,Arg26,34,Phe31-GLP-1(7-36))(Sequence ID 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)(Sequence ID 6);Arg34-GLP-1(7-37)(Sequence ID 7);Glu30-GLP-1(7-37)(Sequence ID 7);Glu30-GLP-1(7-37)(Sequence ID 6) Number 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) This includes analogues of the GLP-1 peptide conjugate moiety, such as 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), and Gly8.36,Glu22,Asn34-GLP-1(7-36) (SEQ ID NO: 21). Any of the aforementioned analogues can be selectively amidated.

[0296] In certain embodiments, the compound includes oligonucleotides, optionally conjugate linkers, and, but is not limited to, analogs of the GLP-1 peptide conjugate moiety, including 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 diabesity,” 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 a 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), where 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 a 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), where Aib is aminoisobutyric acid and Pen is penicillamine.

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

[0300] 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:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 25). 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-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 thereof, the amino acid sequence:AGEGTFTSDVSSYLEGQAAKEAIAWLVKGGPSSGAPPSC (which, in 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 a C-terminal amide.

[0302] 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 (where X is penicillamine) (which in 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 a 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 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 a C-terminal amide.

[0304] 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVK (which, in 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 an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of the amino acid sequence:HAibEGTFTSDVSSYLEEQAAKEFIAWLVK-NH2 (SEQ ID NO: 29), where Aib is aminoisobutyric acid and NH2 represents a 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 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 a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGG-NH2 (SEQ ID NO: 31), where Aib is aminoisobutyric acid and NH2 represents a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGP-NH2, (SEQ ID NO: 32), where Aib is aminoisobutyric acid and NH2 represents a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPS-NH2 (SEQ ID NO: 33), where Aib is aminoisobutyric acid and NH2 represents a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSS-NH2 (SEQ ID NO: 34), where Aib is aminoisobutyric acid and NH2 represents a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSG-NH2 (SEQ ID NO: 35), where Aib is aminoisobutyric acid and NH2 represents a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGA-NH2 (SEQ ID NO: 36), where Aib is aminoisobutyric acid and NH2 represents a 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 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)), 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:HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAP-NH2 (SEQ ID NO: 37), where Aib is aminoisobutyric acid and NH2 represents a 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 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-Zaa (SEQ ID NO: 38)), where Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa represents [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[0317] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting thereof, the amino acid sequence:HAibEGTFTSDVSSYLEGQAANXEFIAWLVRGRG (which, in 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)), where Aib is aminoisobutyric acid, and X or Xaa is of formula: [ka] It is a lysine (5-azidopentanoamide) that contains [a specific compound].

[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 a C-terminal amide, and X or Xaa is of the formula: [ka] It is a lysine (5-azidopentanoamide) that contains [a specific compound].

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 conventional three-letter notation, 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)), where Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa represents [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

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

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

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

[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 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-Cys (SEQ ID NO: 47)), where 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 a 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 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-Zaa (SEQ ID NO: 48)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

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

[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 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-Zaa (SEQ ID NO: 49)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

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

[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 conventional three-letter notation, 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

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

[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 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-Zaa (SEQ ID NO: 51)), where Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa represents [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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-Zaa (SEQ ID NO: 52)), where Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa represents [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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-Zaa (SEQ ID NO: 53)), where Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa represents [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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-Zaa (SEQ ID NO: 54)), wherein Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa represents [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 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-Zaa (SEQ ID NO: 55)), wherein Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

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

[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 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)), where Aib is aminoisobutyric acid, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[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 a C-terminal amide, and Z or Zaa is [ka] It is 4-azidonorleucine, which contains [the specified compound].

[0351] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting thereof an amino acid sequence:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC (which, in 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 a C-terminal amide.

[0353] In certain embodiments, the compound comprises 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 acid moieties that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotide and the isolength portion of any one amino acid sequence 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 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 acid moieties 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 oligonucleotide, a conjugate linker, and an isolength portion of any one of the amino acid sequences of SEQ ID NOs: 1 to 57.

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

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

[0356] In certain embodiments, the compound comprises a GLP-1 peptide conjugate moiety containing an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or combinations thereof, when compared with the amino acid sequence of the oligonucleotide and GLP-1(7-37)(SEQ ID NO: 1).

[0357] In certain embodiments, the compound comprises an oligonucleotide, optionally a conjugate linker, and a GLP-1 peptide conjugate moiety comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1 to 57. In certain embodiments, the compound comprises 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 two or more combinations thereof when compared with the oligonucleotide and one of the amino acid sequences of SEQ ID NOs: 1 to 57. In certain embodiments, the compound comprises 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 two or more combinations thereof when compared with the oligonucleotide, a conjugate linker, and one of the amino acid sequences of SEQ ID NOs: 1 to 57.

[0358] In any of the embodiments described above, the GLP-1 peptide conjugate moiety may include a conservative amino acid substitution, an amino acid analog, or an amino acid derivative. In a particular embodiment, a conservative amino acid substitution includes the substitution of an aliphatic amino acid with another aliphatic amino acid; the substitution of serine with threonine or vice versa; the substitution of an acidic residue with another acidic residue; the substitution of an amide-supporting residue with another amide-supporting residue; the exchange of a basic residue with another basic residue; or the substitution of an aromatic residue with another aromatic residue, or a combination thereof, wherein the aliphatic residue includes alanine, valine, leucine, isoleucine, or synthetic equivalents thereof; the acidic residue includes aspartic acid, glutamic acid, or synthetic equivalents thereof; the amide-containing residue includes aspartic acid, glutamic acid, or synthetic equivalents thereof; the basic residue includes lysine, arginine, or synthetic equivalents thereof; or the aromatic residue includes phenylalanine, tyrosine, or synthetic equivalents thereof.

[0359] Further GLP-1 peptide conjugate portions or analogues 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; International Publication No. 2007 / 124461; International Publication No. 2014 / 096179; International Publication No. 2009 / 030738; International Publication No. 2016 / 055610; and U.S. Patent No. 8,329,419 (all of which are incorporated herein by reference as a whole).

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

[0361] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups 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 bonding group.

[0362] In certain embodiments, conjugate linkers, for example, the conjugate linkers described herein, are known to be useful for attaching conjugate groups to difunctional bonding moieties, such as parent compounds, such as oligonucleotides provided herein. Generally, a difunctional bonding moiety comprises at least two functional groups. One functional group is selected to bond to a specific site of the compound, and the other is selected to bond to a conjugate group. Examples of functional groups used in a difunctional bonding moiety include, but are not limited to, electrophiles for reacting with nucleophiles and nucleophiles for reacting with electrophiles. In certain embodiments, the difunctional bonding moiety comprises one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl groups.

[0363] Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-meleiimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted or unsubstituted C1-C11. 10 Alkyl, substituted, or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Examples of alkynyl substituents include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl substituents.

[0364] In certain embodiments, the conjugate linker contains 1 to 10 linker-nucleosides. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments, such linker-nucleosides contain a modified sugar moiety. In certain embodiments, the linker-nucleosides are unmodified. In certain embodiments, the linker-nucleosides optionally contain a protected heterocyclic base selected from purines, substituted purines, pyrimidines, or substituted pyrimidines. 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 that the linker-nucleosides be cleaved from the compound after reaching the target tissue. Therefore, linker-nucleosides are generally bonded to each other via cleavable bonds and to the rest of the compound. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0365] In this specification, linker nucleosides are not considered part of oligonucleotides. Therefore, in embodiments where a compound comprises a specific number or range of linked nucleosides and / or an oligonucleotide with a specific percentage complementarity to a reference nucleic acid, and the compound also comprises a conjugate group containing a conjugate linker with linker nucleosides, the linker nucleosides are not counted toward the length of the oligonucleotide and are not used to determine the percentage 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 containing 1 to 10 linker nucleosides consecutive to the nucleosides of the modified oligonucleotide. The total number of consecutive linked nucleosides in such a compound may exceed 30. Alternatively, a compound may comprise a modified oligonucleotide consisting of 8 to 30 nucleosides and not include a conjugate group. The total number of consecutive linked nucleosides in such a compound may be 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-nucleosides.

[0366] In certain embodiments, it is desirable that the conjugate group be cleaved from the oligonucleotide. For example, in certain circumstances, a compound containing a particular conjugate moiety is better absorbed by a particular cell type, but when the compound is absorbed, it is desirable that the conjugate group be cleaved to deconjugate or release the parent oligonucleotide. Therefore, a particular conjugate 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 an atomic group containing at least one cleavable bond. In certain embodiments, the cleavable moiety contains an atomic group having one, two, three, four, or five or more cleavable bonds. In certain embodiments, the cleavable moiety is selectively cleaved within an intracellular 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 amides, esters, ethers, one or both phosphodiesters, phosphate esters, carbamates, or disulfides. 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 an oligonucleotide and a 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, one or more linker-nucleosides are bonded to each other and / or to the rest of the compound via cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside attached to either the 3' or 5'-terminal nucleoside of the oligonucleotide by phosphate-nucleoside bonds and covalently bonded to the conjugated linker or the rest of the conjugated moiety by phosphate or phosphorothioate bonds. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.

[0369] 1. Specific hexyl amino linkers In a particular embodiment, the compound has the following structure: [ka] (In the formula, each n is independently selected from 0, 1, 2, 3, 4, 5, 6, or 7) It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0370] In a particular embodiment, the compound has the following structure: [ka] (In the formula, each n is independently between 1 and 20; and p is between 1 and 6.) It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0371] In a particular embodiment, the compound has the following structure: [ka] (In the formula, each n is independently between 1 and 20.) It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0372] In a particular embodiment, the compound has the following structure: [ka] (In the formula, each n is independently between 1 and 20.) It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0373] In a particular embodiment, the compound has the following structure: [ka] It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0374] In a particular embodiment, the compound has the following structure: [ka] It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0375] In a particular embodiment, the compound has the following structure: [ka] It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker selected from the following.

[0376] In a particular embodiment, the compound has the following structure: [ka] It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker having [a specific component].

[0377] In a particular embodiment, the compound has the following structure: [ka] (In the ceremony X binds directly or indirectly to the GLP-1 receptor ligand conjugate moiety; and Y binds directly or indirectly to the modified oligonucleotide. It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker having [a specific component].

[0378] In certain embodiments, the compound comprises an oligonucleotide conjugated to a GLP-1 receptor ligand conjugate moiety by any conjugate linker described in International Publication No. 2014 / 179620 (which is incorporated herein by reference in its entirety).

[0379] 2. Specific alkyl phosphate linkers In a particular embodiment, 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 expressed by the formula: [ka] It is a pyrrolidinyl group having; j is either 0 or 1; n is approximately 1 to approximately 10; p is between 1 and approximately 10; m is 0 or 1-4; and If Y is an amino acid, then m is 1. It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker having [a specific component].

[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 compound 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 a particular embodiment, the compound has the following structure: [ka] (In the formula, X is attached directly or indirectly to the GLP-1 receptor ligand conjugate portion; and T1 contains a modified oligonucleotide; and Bx is a modified or unmodified nucleic acid base. It contains an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker having [a specific component].

[0383] 3. Specific click chemistry linker In certain embodiments, the compound comprises an oligonucleotide conjugated to a GLP-1 receptor ligand conjugate by a conjugate linker, the conjugate linker being prepared using click chemistry known in the art. The compound is prepared using click chemistry, in which the alkynylphosphonate nucleoside bond on the oligomeric compound attached to a solid support is converted to a 1,2,3-triazolylphosphonate nucleoside bond, and then cleaved from the solid support (Krishna et al., J.Am.Chem.Soc. 2012, 134(28), 11618-11631) (this is incorporated herein by reference in its entirety). Further 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 (this is incorporated herein by reference in its entirety).

[0384] In a particular embodiment, using a click reaction, [ka] This is not limited to, but includes the following compounds: [ka] (In the formula, Y attaches directly or indirectly to the oligonucleotide.) Reacting with an oligonucleotide having a terminal amine, [ka] This is obtained and reacted with a GLP-1 receptor ligand conjugate moiety containing an azide, [ka] (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the rest of the GLP-1 receptor ligand conjugate moiety.) By obtaining this, the GLP-1 receptor ligand conjugate portion can be bound to the oligonucleotide.

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

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

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

[0388] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (wherein NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the rest of the GLP-1 receptor ligand conjugate moiety; and Y attaches to oligonucleotides directly or indirectly. Includes.

[0389] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (wherein NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the rest of the GLP-1 receptor ligand conjugate moiety; and Y attaches to oligonucleotides directly or indirectly. Includes.

[0390] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (wherein NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is attached directly or indirectly to the rest of the GLP-1 receptor ligand conjugate moiety; and Y attaches to oligonucleotides directly or indirectly. Includes.

[0391] In certain embodiments, the compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion by a conjugate linker, the conjugate linker being prepared using click chemistry and disulfide bonds.

[0392] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the rest of the GLP-1 receptor ligand conjugate moiety; n and o are independently selected from 2 to 10; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0393] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the rest 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 attaches directly or indirectly to oligonucleotides. Includes.

[0394] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the rest of the GLP-1 receptor ligand conjugate moiety; m is 0 or 1; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0395] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the rest of the GLP-1 receptor ligand conjugate moiety; m is 1; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0396] In certain embodiments, the compound comprises an oligonucleotide bound to the GLP-1 receptor ligand conjugate portion by a conjugate linker, and the compound is [ka] (In the formula, NN=N represents the azide group of the GLP-1 receptor ligand conjugate moiety, and X is directly or indirectly attached to the rest of the GLP-1 receptor ligand conjugate moiety; n and o are independently selected from 2 to 10; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0397] 4. Specific maleimides and maleimide acid linkers In certain embodiments, the compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion by a conjugate linker, the conjugate linker is [ka] Includes, X attaches directly or indirectly to the GLP-1 receptor ligand conjugate; and Y attaches to oligonucleotides directly or indirectly.

[0398] In certain embodiments, the above-described conjugate linker can bind a peptide to an oligonucleotide. In certain embodiments, the compound comprises an oligonucleotide bound to a peptide by a conjugate linker, and the conjugate linker is [ka] Includes, X attaches to the peptide directly or indirectly; and Y attaches to oligonucleotides directly or indirectly.

[0399] In certain embodiments, the compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion, such as a peptide, by a conjugate linker, and the conjugate linker is [ka] (In the formula, R=(CH2) n And n is between 1 and 12; X attaches directly or indirectly to the GLP-1 receptor ligand conjugate portion of peptides, etc.; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0400] In certain embodiments, the compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion, such as a peptide, by a conjugate linker, and the conjugate linker is [ka] (In the formula, [ka] and; m is between 1 and 12; X attaches directly or indirectly to the GLP-1 receptor ligand conjugate portion of peptides, etc.; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0401] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, the first compound comprising an oligonucleotide conjugated to a GLP-1 receptor ligand conjugate portion such as a peptide by a conjugate linker, the conjugate linker is [ka] (In the formula, R=(CH2) n And n is between 1 and 12; X attaches directly or indirectly to the GLP-1 receptor ligand conjugate portion of peptides, etc.; and Y attaches directly or indirectly to oligonucleotides. The first compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion such as a peptide by a conjugate linker, and the second compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion such as a peptide by a conjugate linker, [ka] (In the formula, R=(CH2) n And n is between 1 and 12; X attaches directly or indirectly to the GLP-1 receptor ligand conjugate portion of peptides, etc.; and Y attaches directly or indirectly to oligonucleotides. Includes.

[0402] In certain embodiments, the composition comprises or consists of a substantially pure mixture of two compounds, the first compound comprising an oligonucleotide conjugated to a GLP-1 receptor ligand conjugate portion such as a peptide by a conjugate linker, the conjugate linker is [ka] (In the formula, [ka] and; m is between 1 and 12; X attaches directly or indirectly to the GLP-1 receptor ligand conjugate portion of peptides, etc.; and Y attaches directly or indirectly to oligonucleotides. The first compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion such as a peptide by a conjugate linker, and the second compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate portion such as a peptide by a conjugate linker, [ka] (In the formula, [ka] And m is between 1 and 12; X attaches directly or indirectly to the GLP-1 receptor ligand conjugate portion of peptides, etc.; and Y attaches directly or indirectly to oligonucleotides. Includes.

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

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

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

[0406] 5. Specific disulfide bonds In certain embodiments, the compound comprises an oligonucleotide bound to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker comprising 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 that attaches directly to the GLP-1 peptide conjugate moiety by a disulfide bond without having a conjugate linker.

[0407] In certain embodiments, the compound includes a linker between the oligonucleotide and the activated disulfide moiety. In other embodiments, 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 methanethiosulfonates and dithiocarbomethoxy. In further embodiments, the activated disulfide is a substituted or unsubstituted dithiopyridyl, a substituted or unsubstituted dithiobenzothiazolyl, or a 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 is of formula -SS(O) n -R1 is present, in the formula, n is 0, 1 or 2; and R1 is selected from substituted or unsubstituted heterocyclic, substituted or unsubstituted aliphatic or -C(O)O-R2, and R2- is substituted or unsubstituted aliphatic.

[0409] In another embodiment, the activated disulfide moiety is of the formula -S-S(O)2-substituted or unsubstituted C1-C 12 alkyl or -S-S-C(O)O-substituted or unsubstituted C1-C 12 alkyl. 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 another embodiment, the divalent linking group has the formula -Q1-G-Q2-, wherein Q1 and Q2 are independently absent or substituted or unsubstituted C1-C 12 alkylene, substituted or unsubstituted arylene or -(CH2) m -O-(CH2) p -, and each m and p are independently integers 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,

Chemical formula

[0412] In certain embodiments, the compound comprises an oligonucleotide bonded 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).

[0413] In certain embodiments, any of the above compounds comprising an oligonucleotide directly or by a conjugate linker described herein, linked to a GLP-1 peptide conjugate moiety by a disulfide bond, may include a disulfide bond between the cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid portion of the GLP-1 peptide conjugate moiety and the oligonucleotide or conjugate linker. In certain embodiments, the compound comprises an oligonucleotide directly linked to a 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 portion 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 to the GLP-1 peptide conjugate moiety, and the oligonucleotide is attached to the conjugate linker. In certain embodiments, the compound comprises an oligonucleotide, a conjugate linker, and a GLP-1 peptide conjugate moiety, wherein a disulfide bond connects the conjugate linker to the cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid portion of the GLP-1 peptide conjugate moiety, and the oligonucleotide is attached to the conjugate linker. In certain embodiments, the cysteine, penicillamine, homocysteine, mercaptopropionic acid, or β-mercapto-β,β,-cyclopentamethylenepropionic acid moiety is located 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 bound to a GLP-1 receptor ligand conjugate moiety by a conjugate linker, the conjugate linker comprising an enzymatically cleavable moiety. In certain embodiments, the GLP-1 receptor ligand conjugate moiety is a GLP-1 peptide conjugate moiety. In certain embodiments, the enzymatically cleavable moiety is a peptide such as a dipeptide.

[0415] Enzymes known in the art used to activate prodrugs can be used to cleave the enzymatically cleavable moiety provided in a particular embodiment. In a particular embodiment, the enzymatically cleavable moiety may be cleaved by DT diaphorase, plasmin, carboxypeptidase G2, thymidine kinase (virus), 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 enzymatically cleavable portion can be cleaved by a protease or peptidase. In certain embodiments, the enzymatically cleavable portion is gastrixin, memapsin-2, chymosin, renin, renin-2, cathepsin D, cathepsin E, penicillopepsin, rhizopspepsin, mucorpepsin, barrierpepsin, aspergylopepsin I, endothiapepsin, saccharopepsin, phytepsin, plasmmepsin-1, plasmmepsin-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, mouse leukemia virus type retropepsin, Mason-Pfizer leukemia virus retropepsin, human endogenous retropepsin Illus K retropepsin, retropepsin (human T-cell leukemia virus), bovine leukemia virus retropepsin, Rous sarcoma virus retropepsin, scytalido glutamate peptidase, aspergilloglutamate peptidase, thermopsin, signal peptidase II, spumapepsin, type 4 prepyrine peptidase 1, omptin, plasminogen activator Pla, papain, chymopapain, calicaine, glycylendopeptidase, stem bromelain, ficaine, actinidine, cathepsin V, vignain, cathepsin X, zingipain, cathepsin F, ananaine, fruit bromelain, cathepsin L, cathepsin L1 (Fasciola sp.)), Cathepsin S, Cathepsin K, Cathepsin H, Allurein, Historisine, Cathepsin B, Dipeptidyl-peptidase I, Peptidase 1 (Mite), CPB Peptidase, Kurujipain, 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 Picornine 3C,Hepatitis A virus type picornaine 3C, human rhinovirus type 2 picornaine 3C, foot-and-mouth disease virus picornaine 3C, enterovirus picornaine 2A, rhinovirus picornaine 2A, nuclear inclusion-a peptidase (plum poxvirus), tobacco etch virus NIa peptidase, adenine, potato virus type Y helper component peptidase, Sindbisvirus type nsP2 peptidase, streptopine, clostripine, ubiquitinyl hydrolase-L1, ubiquitinyl hydrolase-L3, regmain ( Plant β-type, Regmine, 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, Timovirus peptidase, Rabbit hemorrhagic disease virus 3C-like peptidase, Pingipain RgpA, Pingipain Kgp, γ-Glutamylhydrase, Foot-and-mouth disease virus L-peptidase, Porcine infectious gastroenteritis virus Illus-type main peptidase, calicivirin, stafopain A, Ulp1 peptidase, separase (yeast type), YopJ protein, PfpI peptidase, saltase A (Staphylococcus type), aminopeptidase N, lysylaminopeptidase (bacteria), aminopeptidase A, leukotriene A4 hydrolase, pyroglutamyl-peptidase II, cytozolaranylaminopeptidase, cystinylaminopeptidase, aminopeptidase B, aminopeptidase Ey, Angiotensin-Converting Enzyme Peptidase Unit 1, Peptidyl-Dipeptidase Acer, Angiotensin-Converting Enzyme Peptidase Unit 2, Angiotensin-Converting Enzyme-2, Timet-Oligopeptidase, Neurolysin, Saccharolisin, Oligopeptidase A, Peptidyl-Dipeptidase Dcp, Mitochondrial Intermediate Peptidase, Oligopeptidase F, Thermolysin, Vibriolisin, Pseudolysin, Coccolisin, Aureolisin, Stearollysin, Mycolysin, Snaparisin,Reishimanorisin, 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 (Homo (sapiens type), Matrix Metallopeptidase-11, Matrix Metallopeptidase-7, Matrix Metallopeptidase-12, Embellicin, Matrix Metallopeptidase-13, Membrane-type Matrix Metallopeptidase-1, Membrane-type Matrix Metallopeptidase-2, Matrix Metallopeptidase-20, Fragilysin, Matrix Metallopeptidase-26, Ceralysin, Aeruginolysin, Gametolysin, Astacin, Meprin α-subunit, Procollagen C-peptidase, Coliolisin L, coriolisin H, flavastacin, fibrolase, jarralagin, adamaricin, atrolisin A, atrolisin B, atrolisin C, atrolisin 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, pitrilysin, inslysin, 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-soluble metallopeptidase, staphylolisin, lysostaffin, methionyl aminopeptidase 1 (Escherichia type), methionyl aminopeptidase 2, Xaa-Pro dipeptidase (bacterial type), aminopeptidase P (bacteria), aminopeptidase P2, Xaa-Pro dipeptidase Eukaryotes, IgA1-specific metallopeptidase, tentoxylicin, vontoxylicin, aminopeptidase Y, aminopeptidase Ap1, aminopeptidase S (Streptomyces type), glutamate carboxypeptidase II, carboxypeptidase Taq, lethal anthrax factor, deuterolysin, peptidyl-Lys Talopeptidase, FtsH peptidase, m-AAA peptidase, i-AAA peptidase, AtFtsH2 peptidase, Paparicin-1, Ste24 peptidase, Dipeptidyl-peptidase III, Site 2 peptidase, Spore formation factor SpoIVFB, HybD peptidase, gpr peptidase, Chymotrypsin A (bovine type), Granzyme B (human (Homo (Sapiens type), Factor VII-Activated Peptidase, Trypsin (Streptomyces griseus type), Hypodermin C, Elastase-2, Cathepsin G, Myeloblastin, Granzyme A, Granzyme M, Chymase (Homo sapiens type), Mast Cell Peptidase 1 (Rattus type), Duodenaise, 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 (brown rat (Rattus norvegicus)), kallikrein 13 (house mouse (Mus musculus)), Ankrod, botrombin, complement factor D, complement component activated C1r, complement component activated C1s, complement factor Bb, mannan-binding lectin-related serine peptidase 1, complement factor I, coagulation factor XIIa, plasma kallikrein, coagulation factor XIa, coagulation factor IXa, coagulation factor VIIa, coagulation factor Xa, thrombin, protein C (activated), coagulation factor C (horseshoe crab (Limulus), horseshoe crab (Tachypleus)), activated, coagulation factor B (ca Horseshoe crab (Limulus), horseshoe crab (Tachypleus), activation, coagulation enzyme (horseshoe crab (Tachypleus) type), acrosin, hepsin, mannan-binding lectin-related serine peptidase 2, urokinase-type plasminogen activator, t-plasminogen activator, plasmin, kallikrein-related peptidase 6, plasminogen activator (vampire bat (Desmodus) type), kallikrein-related peptidase 8, Kallikrein-related peptidase 4, streptoglycin A, streptoglycin B, streptoglycin E, α-soluble endopeptidase, glutamyl peptidase I, DegP peptidase, HtrA2 peptidase, lysyl endopeptidase (bacteria), kallikrein-related peptidase 7, matryptase, togavirin, IgA1-specific serine peptidase (Neisseria type), flavivirin, subtilisin Carlsberg, subtilisin lentus (lentu s), Thermidase, Subtilisin Ak1, Lactosepin I, C5a Peptidase, Dentilysin, Subtilisin BPN', Subtilisin E, Aquarisin 1, Cerevicin, Orijen, Endopeptidase K, Thermomycoline, 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 (eukaryotes), acylaminoacyl peptidase, fibroblast-activating 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, assembryn, cytomegalovirus assembryn, herpesvirus type 8 assembryn, repressor LexA, UmuD protein Signal peptidase I, mitochondrial inner membrane peptidase 1, signal peptidase SipS, signalase (animal) 21kDa component, lysosomal Pro-Xaa carboxypeptidase, dipeptidyl-peptidase II, hepasibirin, potivirus P1 peptidase, pestivirus NS3 polyprotein peptidase, equine arteritis virus serine peptidase, prolylaminopeptidase, C-terminal processing peptidase-1, C-terminal processing peptidase-2, trichorne core peptidase (archaea), signal peptide Peptidase A, Infectious Pancreatic Necrotizing Birnavirus Vp4 Peptidase, Dipeptidase E, Sedrisin, Sedrisin-B, Tripeptidyl-Peptidase I, Coumamorisin, 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-Glutamyl Transferase 1 (Bacterial Type), Mulein tetrapeptidase LD-carboxypeptidase (Escherichia type), PepA aminopeptidase, presenilin 1, polyporopepsin, canditropsin, candidapepsin SAP2, caspase-2, caspase DRONC (Drosophila emeranogaster) type peptidase, ubiquitin-specific peptidase 7, human coronavirus 229E main peptidase, SARS coronavirus picornaine 3C-like peptidase, AvrPphB peptidase, saltase B,The material can be cleaved by proteases or peptidases selected from psychrophilic alkaline metallopeptidases (Pseudomonas sp.), acutolysin A, aminopeptidase S (Staphylococcus type), carboxypeptidase Pfu, isoaspartyl dipeptidase (metallotype), D-aminopeptidase DppA, and murein endopeptidase. In certain embodiments, the enzymatically cleavable portion can be cleaved by cathepsin proteases or peptidases.

[0417] Composition and method for formulating a pharmaceutical composition The compounds described herein can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on numerous criteria, including, but not limited to, the route of administration, the severity of the disease, or the dose administered.

[0418] Certain embodiments provide a pharmaceutical composition comprising one or more compounds or salts thereof. In certain embodiments, the pharmaceutical composition comprises the compounds 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 one 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 comprising the compounds described herein include any pharmaceutically acceptable salts, esters, or salts of such esters or any other oligonucleotides that, when administered to animals including humans, provide (directly or indirectly) bioactive metabolites or residues thereof. Specific embodiments show salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents of pharmaceutically acceptable compounds. Preferred pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0420] Non-exclusive disclosure and incorporation by reference While the specific compounds, compositions, and methods described herein are described in terms of specific embodiments, the following examples are merely illustrative of the compounds described herein and are not intended to limit them.

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

[0422] The sequence list accompanying this application identifies each sequence as either "RNA" or "DNA" as necessary, but in practice, these sequences may be modified with any combination of chemical modifications. Those skilled in the art will readily recognize that designations such as "RNA" or "DNA" describing modified oligonucleotides are optional in particular. For example, an oligonucleotide containing a nucleotide with a 2'-OH sugar moiety and a thymine base may be described as DNA with a modified sugar (a 2'-OH instead of one 2'-H in the DNA) or RNA with a modified base (thymine (methylated uracil) instead of uracil in the RNA). Thus, nucleic acid sequences provided herein, including, but not limited to, those included in the sequence list, are intended to encompass nucleic acids, including, but not limited to, any combination of natural or modified RNA and / or DNA, including nucleic acids having such modified nucleic acid bases. As further examples, and without limitation, the oligomeric compounds having the nucleic acid base sequence "ATCGATCG" include any oligomeric compounds having such a nucleic acid base sequence, whether modified or unmodified, and these oligomeric compounds include, but are not limited to, compounds containing RNA bases such as those having the sequence "AUCGAUCG", compounds containing several DNA bases and several RNA bases such as "AUCGATCG", and oligomeric compounds having other modified nucleic acid bases such as "ATmCGAUCG" (where mC represents a cytosine base with a methyl group at position 5).

[0423] The compounds described herein include (R) or (S), such as α or β in the case of sugar anomers or (D) or (L) 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. Similarly, all cis- and trans-isomers as well as tautomers are included. The compounds described herein include chirally pure or enriched mixtures and racemic mixtures. For example, oligonucleotides having multiple phosphorothioate internucleoside linkages include such compounds where the chirality of the phosphorothioate internucleoside linkages is regulated or random.

[0424] Unless otherwise indicated, any compound containing an oligomeric compound described herein includes its pharmaceutically acceptable salts.

[0425] The compounds described herein include variations in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated element. For example, compounds herein containing a hydrogen atom include all possible deuterium substitutions of each 1 H hydrogen atom. Isotope substitutions included by the compounds herein include, but are not limited to, 1 in place of 2 H or 3 H, 12 in place of 13 C or 14 C, 14 in place of 15 N, 16 in place of 17 O or 18 O and 32 in place of 33 S, 34 S, 35 S or 36 S.

Examples

[0426] Example 1: Preparation of MALAT1-targeting antisense oligonucleotide (ASO) conjugated with GLP-1 peptide. A method for preparing a conjugated modified oligonucleotide containing 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 construction blocks and solid supports are used for incorporating nucleoside residues, for example, those containing T, A, G, and mC residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for 2'-deoxyribonucleosides, cEt BNA nucleosides, and suitably protected 6-aminohexanols. [ka] 5'-Hexylamino-modified oligonucleotide (ISIS 786434) (nucleic acid sequence: TCAGCATTCTAATAGCAGC (SEQ ID NO: 38)) was synthesized and purified using a standard solid-phase oligonucleotide procedure. 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). Approximately 6 μmol of the modified oligonucleotide 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 the reaction was allowed to proceed at room temperature. The reaction was completed after 2-3 hours, and product 2 was HPLC on Source 30Q resin using buffer A 100 mM NH4OAc / 30%ACN / H2O and buffer B 100 mM NH4OAc / 30%ACN / H2O + 1.5M The product was purified using NaBr and desalted by HPLC on a reverse-phase column. The product fraction was concentrated and stored at -20°C.

[0428] Compound 2 was used as a starting material for the reaction with the GLP-1 peptide HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGlyProSerSerAlaProProProSerCys-NH2 (SEQ ID NO: 22), which was synthesized by standard solid-phase peptide synthesis. Aib is 2-aminoisobutyric acid. Compound 2 was dissolved in degassed water, and 0.1 M NaHCO3 was added to adjust the pH to approximately 8.0. 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 amounts (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 a Source 30Q resin using buffer A 100mM NH4OAc / 30%ACN / H2O and buffer B 100mM NH4OAc / 30%ACN / H2O + 1.5M NaBr. The product fraction was desalted by HPLC on a reversed-phase column to obtain ISIS 816385.

[0429] Example 2: Preparation of MALAT1-targeting antisense oligonucleotides (ASOs) conjugated with GLP-1 peptide. A method for preparing a conjugated oligonucleotide containing 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 a starting material for a reaction with the GLP-1 peptide: HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGlyProSerSerAlaProProProSerPen-NH2 (SEQ ID NO: 23), which was synthesized by standard solid-phase peptide synthesis. Aib is 2-aminoisobutyric acid and Pen is penicillamine. Compound 2 was dissolved in degassed water and 0.1 M NaHCO3 was added to adjust the pH to approximately 8.0. The GLP-1 peptide was dissolved in degassed water. The solution of Compound 2 and the peptide solution were mixed by gentle vortexing and the pH was checked. 0.1 M NaHCO3 was added to adjust the pH to approximately 7.5. After about 2 hours, additional peptide was added and NaHCO3 was added to adjust the pH upwards. The reaction was carried out at 4°C for approximately 65 hours, and the product was purified by HPLC as described in Example 1.

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

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

[0432] A control 5'-GLP-1 peptide conjugate ASO having a nucleic acid sequence mismatched to ION 913195 and FOXO1 was prepared starting from a 5'-hexylamino modified oligonucleotide (ION 913194) (nucleic acid sequence: TCAGGCCAATACGCCGTCA (SEQ ID NO: 60)) having 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), following the procedure of Example 1.

[0433] Example 4: Preparation of insulin-targeting antisense oligonucleotides (ASOs) conjugated with GLP-1 peptide A method for preparing a conjugated oligonucleotide containing GLP-1 conjugated at the 5' position via a mercaptopropionate linker.

[0434] ION 919553, an insulin-targeting 5'-GLP-1 peptide conjugate ASO, was prepared starting from a 5'-hexylamino modified oligonucleotide (ION 919553) (nucleic acid sequence: TCAGCCAAGGTCTGAAGGTCACC (SEQ ID NO: 61)) having 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) and following the procedure of Example 1.

[0435] Example 5: Preparation of MALAT1-targeting antisense oligonucleotide (ASO) conjugated with GLP-1 peptide ION 951976 (nucleic acid sequence: GCTGCTATTAGAATGC (SEQ ID NO: 62)) having chemical modifications: Ges mCeo Tdo Gdo mCdo Tdo Ado Tdo Tdo Tdo Ado 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 a standard solid-phase oligonucleotide procedure. ISIS 816385 described in Example 1 was hybridized with ION 951976 to produce a double helix of two oligonucleotides.

[0436] Example 6: In vivo targeting of pancreatic β-islet cells using GLP-1 peptide conjugate ASO Research 1 To determine whether GLP-1 peptide conjugation to ASO increases ASO delivery to the pancreas, male C57BL / 6 mice fed solid diet received intravenous injections of either 3-10-3 cEt ASO (ISIS 556089) (nucleotide sequence: GCATTCTAATAGCAGC) (SEQ ID NO: 63), which targets mouse MALAT1, or GLP-1 conjugated MALAT1 ASO (ISIS 816385) as described in Example 1, at concentrations of 1.8, 0.6, or 0.2 μmol / kg. Tissue samples were collected 72 hours after the final injection to evaluate compound delivery and efficacy.

[0437] MALAT1 expression was detected using the QuantiGene ViewRNA 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 tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4 mm sections. After deparaffinization, the tissue slides were boiled in Affymetrix pretreatment solution for 10-30 minutes, and then treated with protease at 40°C for 10-40 minutes depending on the tissue. The MALAT1 RNA probe was used at a 1:40 dilution and incubated with the sample at 40°C for 120 minutes. After washing, the MALAT1 RNA / probe complex was hybridized with the preamplifier, amplifier, and AP oligonucleotide at 40°C for 25, 15, and 15 minutes, respectively. After 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 β-islet cells but not in pancreatic acinar cells. ASO staining of pancreatic sections showed that GLP-1 conjugate improved efficacy by increasing ASO delivery to tissue. Mice treated with GLP-1 conjugated MALAT1 ASO (ISIS 816385) showed reduced MALAT1 expression in pancreatic β-islet cells, but not in mice treated with unconjugated MALAT1 ASO (ISIS 556089). Mice treated with various doses of ISIS 816385 showed reduced MALAT1 expression in pancreatic β-islet cells. Mice treated with GLP-1 conjugated MALAT1 ASO (ISIS 816385) showed ASO accumulation in pancreatic β-islet cells, but mice treated with unconjugated MALAT1 ASO (ISIS 556089) did not. GLP-1 conjugated MALAT1 ASO (ISIS 816385) accumulated in a dose-dependent manner in pancreatic β-islet cells of treated mice.

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

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

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

[0442] Example 7: Antisense inhibition of MALAT1 and FOXO1 using GLP-1 peptide conjugate antisense oligonucleotides in HEK293 cells overexpressing the human GLP-1 receptor. Antisense oligonucleotides designed to target MALAT1 and FOXO1 were conjugated to a glucagon-like peptide-1 receptor peptide agonist (GLP-1 peptide), and their effects on human target gene expression were tested using the HEK293 cell line (hGLP1R-HEK), which exhibits stable constitutive expression of the human GLP-1 receptor.

[0443] hGLP1R-HEK cell lines were generated by expressing hGLP1R in Flp-IN(trademark)293 cells. Cultured hGLP1R-HEK cells were seeded at a density of 30,000 cells / well in 96-well plates and conditioned for approximately 24 hours with physiological saline, 100 nM, or 10 μM of the following: MALAT1-targeting non-conjugate parent antisense oligonucleotide ISIS 556089 or FOXO1-targeting ISIS 776102 (nucleic acid sequence: TCTTCTTAAAATACCC) (SEQ ID NO: 64), or the corresponding GLP-1 peptide-conjugate antisense oligonucleotide (MALAT1-targeting ISIS 816385 or FOXO1-targeting ION 913193). After the processing period, cells were harvested, mRNA was isolated, and measured using a nanadrop UV-Vis spectrophotometer to adjust for total RNA content. MALAT1 or FOXO1 mRNA levels were measured by quantitative real-time PCR and normalized to the mRNA level of the housekeeping gene (RPLP0) in the same sample. Human MALAT1 mRNA levels were measured using gene expression assay HS00273907, and FOXO1 mRNA levels were measured using assay Hs01054576 (Applied Biosystems). The mRNA 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] The data are shown as percentage inhibition of MALAT1 or FOXO1 mRNA compared to untreated control cells. White symbols represent treatment with parental antisense oligonucleotides, while black symbols represent treatment with antisense nucleotides conjugated to the GLP-1 peptide. As shown in Figure 1, in the hGLP1R-HEK cell line, antisense oligonucleotides conjugated to the GLP-1 peptide inhibited MALAT1 or FOXO1 mRNA more potently than parental antisense oligonucleotides.

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

[0446] Cultured hGLP1R-HEK, wild-type HEK293 (WT HEK293), or cells expressing the hGPR40 receptor were seeded at a density of 30,000 cells / well in 96-well plates 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 shown as MALAT1 mRNA levels normalized to the housekeeping gene (RPLP0). White symbols represent treatment with a parent antisense oligonucleotide (ISIS 556089) that targets MALAT1, while black symbols represent treatment with the same antisense nucleotide (ISIS 816385) conjugated to a GLP-1 peptide.

[0447] The half-number inhibitory concentrations (IC50) of each oligonucleotide are shown in the table below.

[0448] [Table 1]

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

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

[0451] Pancreatic islets were isolated by collagenase digestion of the pancreas collected from blood-extracted 12-15 week old female C57BL / 6Crl mice. The islets were maintained in tissue culture until use. The islets were separated into single cells by shaking in a medium containing a low extracellular calcium concentration. 10-20 intact or isolated islets were seeded in plastic petri dishes and treated with 10 μM antisense oligonucleotides for approximately 24 hours. After the treatment period, the cells were harvested, RNA was isolated, and adjusted for total RNA content 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), the reverse sequence CGTAACTTGATTTGCTGTCCTGAA (SEQ ID NO: 69), and the probe sequence TGAGCCCTTTGCCCCAGATGCCTAT (SEQ ID NO: 70). All data were normalized to the mRNA levels of the housekeeping gene (RPLP0) in the same sample, measured using a primer-probe set with the forward sequence GAGGAATCAGATGAGGATATGGGA (SEQ ID NO: 71), the reverse sequence AAGCAGGCTGACTTGGTTGC (SEQ ID NO: 72), and the probe sequence TCGGTCTCTTCGACTAATCCCGCCAA (SEQ ID NO: 73).

[0452] Figure 3 shows data for the levels of MALAT1 or FOXO1 mRNA against the housekeeping gene (RPLP0). Star symbols represent no treatment for MALAT1 (ISIS 816385) or FOXO1 (ISIS 919193), white circles represent treatment with parental unconjugated antisense oligonucleotides (ISIS 556089 targeting MALAT1 or ISIS 776102 targeting FOXO1), white squares represent treatment with a scrambled FOXO1 antisense oligonucleotide sequence conjugated to a GLP1 peptide (ION 913195), while black symbols represent treatment with an antisense oligonucleotide conjugated to a GLP1 peptide.

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

[0454] Pancreatic islets were isolated by collagenase digestion of the pancreas collected from euthanized 12-15 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, and harvested after approximately 24, 48, or 96 total treatment times, 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 BCA assay reagent (Pierce). FoxO1 protein was detected by Western blot analysis using the primary antibody C29H4 (Cell Signalling, #2880) against FoxO1. α-tubulin was measured using Sigma's primary antibody (#T6074) as a control of the sample loaded onto the gel. For the anti-FoxO1 antibody, the secondary antibody was HRP-conjugate polyclonal goat anti-rabbit P0448 (DAKO), and for the anti-α-tubulin antibody, the secondary antibody was HRP-conjugate polyclonal goat anti-mouse P0447 (DAKO). Enhanced chemiluminescence reagent (Pierce) was used for detection.

[0455] Inhibition of FOXO1 mRNA is shown as FOXO1 mRNA against housekeeping genes, expressed as a percentage of untreated cells in the table below. This shows a slight decrease in mRNA with unconjugated antisense oligonucleotide (ISIS 776102) and a decrease of over 70% with GLP-1 conjugated antisense oligonucleotide (ION 913193).

[0456] [Table 2]

[0457] Western blotting showed a decrease in FoxO1 protein levels measured in pancreases treated with vehicle or antisense oligonucleotides for 96 hours. Protein levels were quantified by measuring the intensity of 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: Incorporation of antisense oligonucleotides into in situ islets of Langerhans in the pancreas after 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 evaluate the uptake of antisense oligonucleotides into pancreatic islets after intravenous or subcutaneous administration of the treated material.

[0459] Female C57BL / 6Crl mice were assigned to five treatment groups. Two groups received either a vehicle (saline) or 2 μmol / kg GLP-1 conjugate antisense oligonucleotide (ISIS 816385) via tail vein injection. Three groups received either saline, 2 μmol / kg unconjugate parental antisense oligonucleotide (ISIS 556089), or 2 μmol / kg GLP-1 conjugate antisense oligonucleotide (ISIS 816385) via subcutaneous administration twice weekly for two weeks. All animals were sacrificed approximately 72 hours after the final dose, and the pancreas was collected for ex vivo analysis by immunohistochemical examination of antisense oligonucleotide uptake.

[0460] All tissues were fixed in 10% neutral buffered formalin at room temperature for 32 hours. After fixation, samples were dehydrated using a standard series of ethanols followed by xylene and embedded in paraffin. Tissue sections were cut to a thickness of 4 μm, placed on superfrost® Plus slides, and baked in a drying oven at 60°C for 1 hour. Immunohistochemical testing for antisense oligonucleotide detection was performed in a Ventana Discovery XT immunohistochemistry machine (Ventana Medical System, Inc.) according to manufacturer recommendations, with all reagents being Ventana products (Roche Diagnostics, Basel, Switzerland). Protease 1 was used as the enzyme antigen retrieved, and the incubation was 8 minutes. After adding an antibody blocker for 4 minutes to reduce background, rabbit anti-ASO 2.5 was added at 37°C for 1 hour (dilution 1:5000, Ionis Pharmaceuticals). For secondary detection, OmiMap anti-rabbit HRP was incubated for 16 minutes, followed by color detection using the DISCOVERY ChromoMap DAB kit (RUO). The slides were counterstained with hematoxylin for 4 minutes, and then blued for 4 minutes. The stained slides were analyzed under a standard bright-field microscope.

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

[0462] Example 12: Antisense inhibition of MALAT1 in the in situ islets of Langerhans in the pancreas after administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotide in C57BL / 6Crl mice. Unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides against MALAT1 were further tested in vivo to evaluate antisense inhibition of MALAT1 in the pancreas after intravenous or subcutaneous administration of the 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 the pancreas was harvested for ex vivo analysis of MALAT1 expression by in situ hybridization.

[0464] Tissue was prepared as described herein (Example 11). In-situ mRNA amplification and labeling processes were performed on the Ventana Discovery ULTRA, Automated ISH platform (Ventana Medical System, Inc.) using the RNAscope® VS assay based on Advanced Cell Diagnostics (ACD). A custom-ordered probe was obtained from ACD for the detection of MALAT1 mRNA, and various parameters were tested to optimize a novel RNAscope method for ISH. The signal was amplified using multiple steps, followed by probe labeling and detection using the RNAscope® 2.5 VS Reagent Kit-RED. Stained slides were analyzed under a standard bright-field microscope.

[0465] MALAT1 expression was decreased in the pancreatic islets of Langerhans of animals treated subcutaneously or intravenously with GLP-1 peptide-conjugated antisense oligonucleotide (ISIS 816385), but not in exocrine tissues. MALAT1 expression was not decreased in animals subcutaneously treated with unconjugated parental 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. Unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides to MALAT1 were further tested in vivo to evaluate the hepatic uptake of antisense oligonucleotides via either intravenous or subcutaneous administration.

[0467] The animals were assigned to the treatment as described herein (Example 11). All animals were sacrificed approximately 72 hours after the final dose, and the pancreas was recovered for ex vivo analysis by immunohistochemical examination of antisense oligonucleotide uptake.

[0468] Tissue was prepared and immunohistochemical analysis was performed as described herein (Example 12).

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

[0470] Example 14: Antisense inhibition of MALAT1 in the liver after administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotide 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 via 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 of treatment, and the livers were collected for ex vivo analysis of MALAT1 expression by in situ hybridization.

[0472] Tissue was 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 higher degree than in hepatic parenchymal cells of animals treated with ISIS 556089 via subcutaneous administration. 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 of Langerhans and liver 72 hours after administration of a single dose of unconjugated parental and GLP-1 peptide-conjugated antisense oligonucleotides in C57BL / 6Crl mice. Unconjugated parent molecules and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to evaluate the efficacy of MALAT1 antisense inhibition in isolated pancreatic islets of Langerhans in the liver 72 hours after a single subcutaneous administration.

[0475] Female C57BL / 6Crl mice were assigned to eight treatment groups, receiving 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, while 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 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 (control).

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

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

[0478] Female C57BL / 6Crl mice were assigned to seven treatment groups, receiving 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, while two treatment groups received either 0.01 μmol / kg or 1 μmol / kg ISIS 776102. All animals were sacrificed 72 hours after the final dose. For mRNA analysis, liver samples were collected and pancreatic islets isolated as described herein (Example 9). FOXO1 mRNA levels were quantified as described herein (Example 9) and expressed as a percentage of vehicle-treated animals (control).

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

[0480] Example 17: Antisense inhibition of FOXO1 in isolated islets of Langerhans and liver 6 weeks after repeated administration of unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides to ob / ob mice. Unconjugated parent and GLP-1 peptide-conjugated antisense oligonucleotides were further tested in vivo to evaluate the efficacy of FOXO1 antisense inhibition in isolated pancreatic islets of Langerhans in the liver 6 weeks after 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 once a week for 6 weeks. Approximately 120 hours after the final dose, all animals were sacrificed, and liver samples were collected for mRNA analysis as described herein (Example 9), and pancreatic islets were isolated. FOXO1 mRNA levels were quantified as described herein (Example 9), and mRNA levels were normalized to the housekeeping gene (RPLP0) in each sample.

[0482] Significant antisense inhibition of FOXO1 was not observed in the liver or in the islets of Langerhans treated with either the parent antisense oligonucleotide (ISIS 776102) or a scrambled FOXO1 antisense oligonucleotide sequence conjugated with GLP-1 peptide in any of the treatment groups (ION 913195). Animals treated with GLP-1 peptide-conjugated antisense oligonucleotide (ION 913193) showed reduced FOXO1 mRNA levels in isolated islets of Langerhans at both dose levels tested (a 42% mean decrease in FOXO1 mRNA at 0.03 μmol / kg and a 72% mean decrease in FOXO1 mRNA at 0.1 μmol / kg), demonstrating that GLP-1 peptide conjugation enhances antisense inhibition in pancreatic islets of Langerhans in vivo.

[0483] Example 18: Decrease in FoxO1 protein levels in islets of Langerhans isolated from ob / ob mice treated for 6 weeks with unconjugated parent or GLP-1 peptide-conjugated antisense oligonucleotides. Unconjugated parental 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 the pancreatic islets were isolated as described herein (Example 10) for FoxO1 protein analysis. Random samples were selected from each treatment group and loaded onto each gel so that at least one sample from each treatment group was analyzed on the same gel. FoxO1 protein levels were measured by quantifying the 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 receiving ION 913195.

[0485] Foxo1 protein levels in animals treated with ION 913193 decreased by 57% and 81% compared to ION 913195 when treated with 0.03 μmol / kg and 0.1 μmol / kg, respectively, and decreased by 64% and 36% compared to animals treated with 0.1 μmol / kg ISIS 776102.

[0486] Example 19: Preparation of GLP-1 peptide-conjugated antisense oligonucleotides targeting MALAT1 A 5'-GLP-1 peptide conjugate ASO targeting ION 962963 and MALAT1 was prepared starting from a 5'-hexylamino modified oligonucleotide (ISIS 722061) (nucleic acid sequence: GCATTCTAATAGCAGC (SEQ ID NO: 65)) having 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), according to the procedure of Example 1.

[0487] Example 20: Preparation of MALAT1-targeting antisense oligonucleotides conjugated to GLP-1 peptide via a clicklinker A method for preparing a conjugated oligonucleotide containing GLP-1 conjugated at the 5' position via a clicklinker.

[0488] Preparation of 5'-BCN MALAT-1 targeted oligonucleotide ISIS 791173: 5'-Hexylamino-modified oligonucleotide (ISIS 786434) (nucleic acid sequence: TCAGCATTCTAATAGCAGC (SEQ ID NO: 58)) was synthesized and purified using a standard solid-phase oligonucleotide procedure. 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)nonano-4-in-9-ylmethyl N-succinimidyl carbonate) was obtained from Aldrich. Approximately 1 g of the modified oligonucleotide was dissolved in 5 mL of sodium tetraborate buffer, pH 8.5. 13.4 mg of the BCN-NHS ester was dissolved in 10 mL of DMSO and added to ASO solution, and stirred at room temperature for 4 hours. The reaction mixture was diluted in 1 M NaCl solution and desalted by HPLC on a reverse-phase column.

[0489] Preparation of GLP-1 click-conjugate ASO (ION 1071996) 12 mg of 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 azide-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. Precipitation was observed after 18 hours, and an additional 1 mL of DMF was added. The reaction was continued for a further 5 hours. The product was purified by HPLC on a SAX column using buffer A 100 mM NH4OAc / 30%ACN / H2O and buffer B 1.5 M NaBr / NH4OAc / 30%ACN / H2O, and desalted by HPLC on a reversed-phase column. The product fraction was collected and lyophilized to obtain the expected conjugated ASO, ION 1071996. [ka]

[0490] Example 21: Antisense inhibition of MALAT1 in mouse primary islets of Langerhans after treatment with non-conjugated parent or GLP-1 peptide-conjugated ASO having various linkers. To determine whether the chemical structure of GLP-1 peptide conjugation to ASO affects ASO delivery into the pancreas, male C57BL / 6 mice received intravenous injections of either ISIS 556089 (parent unconjugated ASO), ISIS 816385 (GLP-1 conjugated ASO with disulfide linker and 5'TCA linker), ION 962963 (GLP-1 conjugated ASO with disulfide linker and no 5' nucleotide spacer) or ION 1071996 (GLP-1 conjugated ASO conjugated via click linker) once weekly at a dose of 0.6 μmol / kg / week for three weeks. Tissue was collected 72 hours after the final injection to evaluate compound delivery and efficacy.

[0491] MALAT1 expression was detected as in Example 6. In-situ hybridization analysis showed that MALAT1 expression in β-islet cells was reduced in mice treated with GLP-1 conjugated ASOs (ISIS 816385, ION 962963, and ION 1071996) compared to saline controls, but not in mice treated with unconjugated parental ASOs (ISIS 556089).

[0492] Example 22: Dose-dependent reduction of MALAT-1 expression in LVX-GLP1R cells HEK cells stably expressing FLAG-tagged GLP1R were generated by infecting them with a lentivirus containing FLAG-tagged GLP1R, produced by transfection of 293T cells using pLVX-IRES-Puro (Clontech Laboratories Inc., Mountainview, CA) containing a FLAG-GLP1R insert. Infected cells were selected with puromycin (2 μg / ml), and receptor expression was then analyzed by Western blotting 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 processing period, total RNA was prepared using the RNeasy mini-kit (Qiagen, Valencia, CA, USA), and qRT-PCR was performed using the primer-probe set RTS2739 (forward sequence: AGGCGTTGTGCGTAGAGGAT (SEQ ID NO: 74), reverse sequence: AAAGGTTACCATAAGTAAGTTCCAGAAAA (SEQ ID NO: 75), probe sequence: AGTGGTTGGTAAAAATCCGTGAGGTCGGX (SEQ ID NO: 76)). Briefly, approximately 50 ng of total RNA in 5 μL of water was mixed with a 0.3 μL primer-probe set containing forward and reverse primers (10 μM each), fluorescently labeled probe (3 μM), 0.3 μL of room temperature enzyme mix (Qiagen), 4.4 μL of RNase-free water, and 10 μL of 2× PCR reaction buffer in a 20 μL reaction mixture. StepOne Plus RT-PCR system (Applied Reverse transcription was performed at 48°C for 10 minutes using Biosystems (Phoenix, AZ, USA), followed by 40 cycles of PCR with 20 seconds at 94°C and 20 seconds at 60°C during each cycle. mRNA levels were normalized to the total RNA amount present in each reaction as determined by the Ribogreen assay (Life Technologies), and then normalized to a saline control (100% expression).The results are shown in the table below, demonstrating increased dose-dependent inhibition of MALAT-1 by GLP-1 complexed ASOs with or without a TCA linker (816385).

[0493] [Table 3]

[0494] Example 23: Effect of peptide length and conjugation site on the in vitro activity of GLP-1 conjugated ASO targeting MALAT1 To evaluate the effects of precise peptide sequence, peptide length, and conjugation site 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 a click reaction with a 5-azidopentanoic acid-modified lysine residue (X), as shown below. Other compounds were synthesized from ION 791173 by a click reaction with a C-terminal azidonorleucine (Z), as shown in Example 20 above. [ka]

[0495] LVX-GLP1R cells (as described in Example 22) were seeded at a density of 10,000 cells / well and incubated with seven doses of peptide-conjugated ASO in a 4-fold dilution series. After the treatment period, total RNA was prepared and analyzed as in Example 22 above. The IC50 values ​​are shown in the table below.

[0496] [Table 4]

[0497] Example 24: Preparation of GLP-1 conjugated siRNA targeting PTEN Method for preparing siRNA nucleotide double helix targeting PTEN ISIS 522247 (nucleic acid base sequence TTATCTATAATGATCAGGTAA (SEQ ID NO: 77) and chemically modified Afs Cms Cfo Ufs Cmo Ufs Amo Ufs Amo Afs Umo Gfs Amo Ufs Cms Afs Gms Gfs Ums Aes Ae (Tx=5'-(E)-vinyl P-2'-O-methoxyethyl-thymine, f=2'-α-fluoro-2'deoxyribose, m=2'-O-methylribose, e=2'O-methoxyethylribose, o=phosphodiester; and s=phosphorothioate) and ISIS 522247 (nucleic acid base 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 (same as above)) The nucleic acid sequence 790973 (ACCTGATCATTATAGATAA, sequence number 78) was synthesized and purified using a standard solid-phase oligonucleotide procedure.

[0498] GLP-1 conjugated ION 1055394 was prepared starting from a 5'-hexylamino modified oligonucleotide (ION 1055395) (ACCTGATCATTATAGATAA (SEQ ID NO: 78)) conjugated to a GLP-1 peptide having the sequence HisAibGluGlyThrPheThrSerAspValSerSerTyrLeuGluGluGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGlyProSerSerGlyAlaProProProSerCys, which contains a free N-terminal amine and a C-terminal amide (the nucleic acid base sequence ACCTGATCATTATAGATAA (SEQ ID NO: 78)) and following the procedure of Example 1.

[0499] ISIS 522247 was hybridized with 790973 to produce a double helix of two oligonucleotides. ISIS 522247 was hybridized with 1055394 to produce a double helix of two oligonucleotides.

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

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

[0502] 27.7 mg of peptide DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC-NH2 (SEQ ID NO: 79) was dissolved in 2 mL of DMF:0.1 M NaHCO3 (1:1) (peptide solution). The peptide solution was slowly added to the ASO solution and stirred at room temperature for 30 minutes. The reaction was monitored by LC-MS and stirring was continued for a further 1 hour. The main 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% aqueous acetonitrile solution; buffer B: 1.5 M NaBr in A, 0-60% B in 28 column volume). The fractions containing full-length ASO were pooled together, diluted to a 10% concentration of 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, evaporated, and the expected product was obtained, as confirmed by LC-MS.

[0503] Example 26: Method for preparing a conjugated 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-maleimide propionate (MW 266.21 g / mol) in sodium tetraborate buffer at pH 7 and room temperature to obtain 5'-(3-maleimidyl)propionyl-C6 MALAT1 ASO. A GLP-1 peptide containing a C-terminal cysteinamide ("GLP-1 peptide-cysteinamide", HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2) was dissolved in 0.1 M sodium phosphate, pH 8.5 / DMF and added to 5'-(3-maleimidyl)propionyl-C6 MALAT1 ASO with stirring at room temperature. The product (ION1086699) was formed.

[0504] Example 27: Method for preparing a conjugated oligonucleotide containing GLP-1 conjugated at the 5' position via a disulfide clicklinker (preparation of Ionis-1123478). A 5'-hexylamino modified oligonucleotide (ISIS 786434) targeting MALAT1 was synthesized on a NittoPhase® HL solid support (115 mg, 47 umol) using an AKTA Oligopilot® synthesizer. A 0.1 M solution of 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) were conjugated to a solid support supporting Ion 925727 using a standard solid-phase oligonucleotide procedure. 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 minutes. The reaction mixture was cooled from 45°C 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 thought to stop upon completion of sodium acetate addition. The product was purified by HPLC on a source 30Q resin using buffer A 100 mM NH4OAc / 30%ACN / H2O and buffer B 100 mM NH4OAc / 30%ACN / H2O + 1.5 M NaBr. The pure fraction was desalted on a reverse-phase column by HPLC to obtain the MMT-deprotected oligonucleotide.

[0505] Next, the deprotected oligonucleotide was reacted with 3 eq. of (1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyl N-succinimidyl carbonate (744867 Aldrich) in 1:2 DMSO-sodium tetraborate buffer pH 8.5 at room temperature to obtain 5'-BCN-C6 MALAT1 ASO. The 5'-BCN-modified ASO was then desalted on a reverse-phase column, dried, and redissolved in 2 mL of sodium tetraborate buffer pH 8.5. A GLP-1 peptide containing C-terminal 4-AzidoNorLeu["GLP1 / Ex4 Fusion Seqence-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 hours, 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 a source 30Q resin using buffer A 100 mM NH4OAc / 30%ACN / H2O and buffer B 100 mM NH4OAc / 30%ACN / H2O + 1.5 M NaBr. The product fraction was desalted by reverse-phase HPLC to obtain ION 1123478, which was confirmed by LC-MS analysis. [ka]

[0506] Example 28: Method for preparing a conjugated 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-maleimide propionate (MW 266.21 g / mol) at pH 7 in sodium tetraborate buffer at room temperature to obtain 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. Desalting was performed using reverse-phase HPLC with buffer A: 100 mM NH4OAc in acetonitrile:water 3:7 (v:v) and buffer B: 1.5 M NaBr and 100 mM NH4OAc in acetonitrile:water 3:7 (v:v). A GLP-1 peptide containing a C-terminal cysteinamide ("GLP-1 peptide-cysteinamide", HAibEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2) was dissolved in 0.1 M sodium phosphate, pH 7.0 / 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 acetonitrile:water 3:7 (v:v), Buffer B: 1.5 M NaBr, 50 mM NaHCO3 in acetonitrile:water 3:7 (v:v). The product fraction was pooled and kept at 5°C for 4 days, and desalted on a reverse-phase column by HPLC to obtain 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 ASO affects ASO delivery into the pancreas, female C57BL / 6Crl mice received a single subcutaneous injection of 0.01 μmol / kg vehicle (physiological saline), ISIS 816385 (GLP-1 conjugated ASO with a disulfide linker), ION 1071996 (GLP-1 conjugated ASO conjugated via a click linker), ION 1086699 (GLP-1 conjugated ASO conjugated via a maleimide linker), ION 1123118 (GLP-1 conjugated ASO conjugated via a maleimide acid linker), or ION 1123478 (GLP-1 conjugated ASO conjugated via a disulfide-click linker). Islets were isolated 72 hours after subcutaneous administration to evaluate compound delivery and efficacy.

[0508] MALAT1 expression was measured as in Example 9 and expressed as a percentage of the vehicle-treated animals (control), and compared with expression in islets from mice treated with ISIS 816395. qPCR quantification showed that MALAT1 expression in β-islet cells was significantly further reduced in mice treated with GLP-1 conjugated ASO ION 1086699, ION 1123118, and ION 1123478 compared to ISIS 816385, and similarly reduced in mice treated with ION 1071996.

[0509] [Table 5]

[0510] Example 30: Dose-response 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 GLP-1 peptide conjugation to ASO affects ASO delivery into the pancreas, female C57BL / 6Crl mice (n=6) received single subcutaneous injections of ION 1086699 (GLP-1 conjugated MALAT-1 ASO conjugated via maleimide linker), ION 1123118 (GLP-1 conjugated MALAT-1 ASO conjugated via maleimide acid linker), or ION 1134165 (GLP-1 conjugated MALAT-1 ASO conjugated via disulfide linker) at concentrations of 0.03, 0.01, 0.001, or 0.0003 μmol / kg. A group of mice (n=6) was subcutaneously injected with 0.01 μmol / kg of ISIS 816385 (GLP-1 conjugated MALAT-1 ASO conjugated via a disulfide linker, as described in Example 1). Another group of mice (n=6) was subcutaneously injected with PBS, which served as a control group for comparison with the other groups. Islets were isolated 72 hours after subcutaneous administration to evaluate the delivery and potency of the compound. Percentage inhibition of MALAT1 mRNA levels was quantified as described herein (Example 9) and averaged for each treatment group against PBS-treated animals (control). 0% inhibition reflects no inhibition of MALAT1 mRNA. ND means that no mice were administered the indicated dose.

[0511] [Table 6]

[0512] The results indicate that GLP-1 conjugated MALAT-1 ASO 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 ASO conjugated via a maleimide or disulfide linker.

[0513] Example 31: Effect of linker on food intake in C57BL / 6Crl mice treated with GLP-1 conjugate antisense oligonucleotides Eight mice were administered a single subcutaneous injection of ION 816385 (GLP-1 conjugated MALAT-1 ASO conjugated via a disulfide linker) or ION 1123118 (GLP-1 conjugated MALAT-1 ASO conjugated via a maleimide acid linker) at concentrations of 0.01, 0.03, or 0.1 μmol / kg, as shown in the table below, and their food intake was monitored for 24 hours. Food intake was monitored as a control for eight mice administered subcutaneous injection of PBS. The maleimide acid linker increased the therapeutic window associated with GLP1-induced food intake decline compared to the disulfide linker.

[0514] [Table 7]

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