Lipid conjugates for the delivery of therapeutic agents

Abstract

Disclosed herein are compounds according to Formula (I) comprising PK / PD modulators for delivery of oligonucleotide-based agents, e.g., double stranded RNAi agents, to certain cell types, for example cardiomyocytes, in vivo. The compounds disclosed herein, when conjugated to an oligonucleotide-based therapeutic or diagnostic agent, such as an RNAi agent, can enhance the delivery of the composition to the specified cells being targeted to facilitate the inhibition of gene expression in those cells.

Description

LIPID CONJUGATES FOR THE DELIVERY OF THERAPEUTIC AGENTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority7to United States Provisional Patent Application Serial No. 63 / 676,686, filed on July 29. 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing (in compliance with Standard ST26), which has been submitted in xml format and is hereby incorporated by reference in its entirety7. The xml sequence listing file is named 30752-WO_SeqListing.xml, created July 25, 2025, and is 967,328 bytes in size.FIELD OF THE INVENTION

[0003] The present disclosure relates to lipid conjugates (also referred to herein as lipid PK / PD modulators) for the delivery of oligonucleotide-based agents, e.g., double stranded RNAi agents, to certain cell ty pes (e.g., cardiomyocytes) in vivo, for inhibition of genes that are expressed in those cells.BACKGROUND

[0004] Oligonucleotide-based agents, such as antisense agents and double stranded RNA interference (RNAi) agents, have shown great promise and have the potential to revolutionize the field of medicine and the availability to patients of potent therapeutic treatment options. However, the effective delivery of oligonucleotide-based agents, and double-stranded therapeutic RNAi agents in particular, has long been a challenge in developing viable therapeutic pharmaceutical agents. This is particularly the case when trying to achieve specific and selective delivery of oligonucleotide-based agents to extra-hepatic (i.e., nonhepatocyte) cells.

[0005] While various attempts over the past several years have been made to direct oligonucleotide-based agents to certain extra-hepatic cell types, including skeletal muscle cells, adipocytes, cardiomyocytes, and the like, using, for example, cholesterol conjugates (which is non-specific and has the known disadvantage of distributing to various undesired tissues and organs) and lipid-nanoparticles (LNPs) (which have been frequently reported to have toxicity concerns), to date none have achieved suitable delivery. As a result, thereremains a need for a delivery' vehicle to direct oligonucleotide-based agents, and RNAi agents in particular, to non-hepatocyte cell types.SUMMARY

[0006] Disclosed herein are compounds comprising a lipid PK / PD modulator conjugated (or connected) to an oligonucleotide-based agent. Lipid PK / PD modulator precursors are also disclosed herein.

[0007] One aspect of the invention provides a compound of Formula (I):wherein,R comprises an oligonucleotide-based agent;Li is a linking moiety;L2 is selected from the group consisting of: optionally substituted alkylene, optionally substituted arylene, 2-20 polyethylene glycol (PEG) units optionally interrupted by C(O)NRa, wherein R3 is selected from Ci-Ce alkyl and H, and a bond;Z is selected from the group consisting of: CH (wherein p is 1), CH2 (wherein p is 0), N (wherein p is 1), optionally substituted arylene, C(O)NR (wherein p is 0), heterocyclene, and NR3 (wherein p is 0), wherein R3 is selected from Ci-Cs alkyl and H; p is 0 or 1, as valency permits;Yi and Y2 are each independently selected from the group consisting of: 2-20 polyethylene glycol (PEG) units, optionally substituted alkyl, optionally substituted cycloalkyl, and a bond;Xi and X2 are each independently selected from the group consisting of: C(O), C(O)NR2 and a bond, wherein R2 is Ci-Ce alkyl or H; n and m are each independently an integer from 8 to 20;Wi and W2 are each independently selected from the group consisting of: H, COOH

[0008] In some embodiments, the oligonucleotide-based agent is an RNAi agent.

[0009] A PK / PD modulator is linked to an oligonucleotide-based agent, such as an RNAi agent, to facilitate delivery of the RNAi agent to the desired cells or tissues. PK / PD modulator precursors can be synthetized having reactive moieties, such as maleimide or azido groups, that readily facilitate linkage to one or more linking groups on an RNAi agent. Chemical reaction syntheses to link such PK / PD modulator precursors to RNAi agents are generally known in the art. The terms "‘PK / PD modulator” and “lipid PK / PD modulator” may be used interchangeably herein.

[0010] Any of the PK / PD modulator precursors disclosed herein such as LP161-p can be used as starting materials to link to RNAi agents. The PK / PD modulator precursors may be covalently attached to an RNAi agent using any known method in the art. For example, in some embodiments, maleimide-containing PK / PD modulator precursors may be reacted with a disulfide-containing moiety on the 3’ end of the sense strand.[OH] In some embodiments, one or more PK / PD modulators may be conjugated to RNAi agents described herein. In some embodiments, one, two, three, four, five, six, seven or more PK / PD modulators may be conjugated to RNAi agents described herein.

[0012] PK / PD modulator precursors may be conjugated to RNAi agents using any known method in the art. In some embodiments, PK / PD modulator precursors comprising a maleimide moiety may be reacted with RNAi agents comprising a disulfide linkage to form a compound comprising a PK / PD modulator conjugated to an RNAi agent. The disulfide may be reduced, and added to a maleimide by way of a Michael-Addition reaction. An example reaction scheme is shown below”wherein Rzz comprises an RNAi agent, andindicates a point of connection to any suitable S-S group known in the art. In some instances of the reaction scheme above, pzz' is attached to an alkyl group such as hexyl (CeHn).

[0013] In some embodiments, PK / PD modulator precursors may comprise a sulfone moiety and may react with a disulfide. An example reaction scheme is shown below:wherein Rzz comprises an RNAi agent, andindicates a point of connection to any suitableS-S group known in the art. In some instances of the reaction scheme above, pzz 1is attached to an alkyl group such as hexyl (CeHn).

[0014] In some embodiments, PK / PD modulator precursors may comprise an azide moiety and be reacted with an RNAi agent comprising an alkyne to form a compound comprising a PK / PD modulator conjugated to an RNAi agent according to the general reaction scheme below:wherein Rzz comprises an RNAi agent.

[0015] In some embodiments, PK / PD modulators may be conjugated to the 5’ end of the sense or antisense strand, the 3’ end of the sense or antisense strand, or to an internal nucleotide of RNAi agents. In some embodiments, an RNAi agent is synthesized with a disulfide-containing moiety at the 3’ end of the sense strand, and a PK / PD modulator precursor may be conjugated to the 3’ end of the sense strand using any of the appropriate general synthetic schemes shown above.DETAILED DESCRIPTION

[0016] Definitions

[0017] As used herein, the terms “oligonucleotide” and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodified.

[0018] As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”) means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with theRNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double stranded RNAs (dsRNA). micro RNAs (miRNAs). short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0019] As used herein, the term "lipid" refers to moieties and molecules that are soluble in nonpolar solvents. The term lipid includes amphiphilic molecules comprising a polar, water- soluble head group and a hydrophobic tail. Lipids can be of natural or synthetic origin. Nonlimiting examples of lipids include fatty acids (e.g., saturated fatty acids, mono unsaturated fatty acids, and polyunsaturated fatty’ acids), glycerolipids (e.g.. monoacylglycerols, diacylglycerols, and triacylglycerols), phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine), sphingolipids (e.g., sphingomyelin), and cholesterol esters. As used herein, the term “saturated lipid” refers to lipids that are free of any unsaturation. As used herein, the term “unsaturated lipid” refers to lipids that comprise at least one (1) degree of unsaturation. As used herein, the term “branched lipid” refers to lipids comprising more than one linear chain, wherein each linear chain is covalently attached to at least one other linear chain. As used herein, the term “straight chain lipid” refers to lipids that are free of any branching.

[0020] As used herein, the terms “silence.” “reduce,” “inhibit,” “down-regulate,” or "knockdow n" when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.

[0021] As used herein, the terms “sequence” and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described w ith a succession of letters using standard nomenclature.

[0022] As used herein, a “base,’' “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry. Biotechnology and Medicine, Herdewijn, P. ed. Wiley -V CH. 2008). The synthesis of such modified nucleobases (including phosphorami dite compounds that include modified nucleobases) is known in the art.

[0023] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleobase or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the abi 1 i ty of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) and form a duplex or double helical structure under certain standard conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence.Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0024] As used herein, “perfectly complementary” or “fully complementary" means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0025] As used herein, “partially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0026] As used herein, “substantially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85%. but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of basesin a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0027] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary ” are used with respect to the nucleobase or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a sequence of a target mRNA.

[0028] As used herein, the term “substantially identical” or “substantial identity.” as applied to a nucleic acid sequence means the nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity or more, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. Percentage of sequence identity' is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In some embodiments, sequence identity, as applicable to a particular nucleotide or amino acid sequence, is calculated by a pairwise alignment using the Needleman -Wunsch algorithm using a generally available alignment program, e.g., the Needle (EMBOSS) program. The inventions disclosed herein encompass nucleotide sequences substantially identical to those disclosed herein. In particular embodiments, a nucleic acid sequence is 99% identical to a nucleotide sequence disclosed herein. In particular embodiments, a nucleic acid is 95% identical to a nucleotide sequence disclosed herein. In particular embodiments, an amino acid sequence is 99% identical to a polypeptide sequence disclosed herein. In particular embodiments, an amino acid is 95% identical to a polypeptide sequence disclosed herein.

[0029] As used herein, the terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include the preventative treatment, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0030] As used herein, the phrase “introducing into a cell,” when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell. The phrase “functional delivery ,” means delivering the RNAi agent to the cell in a manner that enables the RNAi agent to have the expected biological activity, e.g., sequence-specific inhibition of gene expression.

[0031] As used herein, the term “isomers'’ refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a “chiral center.”

[0032] As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are present and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.

[0033] As used in a claim herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0034] The person of ordinary' skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art.

[0035] As used herein, the term “linked” or “conjugated” when referring to the connection between two compounds or molecules means that two molecules are joined by a covalent bond or are associated via noncovalent bonds (e.g., hydrogen bonds or ionic bonds). In some examples, where the term “linked” or “conjugated” refers to the association between two molecules via noncovalent bonds, the association between the two different molecules has a KD of less than 1 x 10’4M (e.g., less than 1 x 10'5M, less than 1 x 10'6M, or less than 1 x 10'7M) in physiologically acceptable buffer (e.g., buffered saline). Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a firstcompound and a second compound either with or without any intervening atoms or groups of atoms.

[0036] As used herein, a linking group is one or more atoms that connects one molecule or portion of a molecule to a second molecule or second portion of a molecule. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with a linking group. Linking groups may comprise any number of atoms or functional groups. In some embodiments, linking groups may not facilitate any biological or pharmaceutical response, and merely serve to link two biologically active molecules.

[0037] As used herein, the term “alkyd” refers to a saturated aliphatic hydrocarbon group containing 1-12 (e.g., 1-8, 1-6, 1-4, or 1-3) carbon atoms. An alkyl group can be straight or branched. Examples of alkyl groups include, but are not limited to. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-buty l, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl.

[0038] Unless stated otherwise, use of the symbolas used herein means that any group or groups may be linked (or connected) thereto that is in accordance with the scope of the inventions described herein.

[0039] As used herein, the term “including” is used to herein mean, and is used interchangeably with, the phrase “including but not limited to.” The term “or” is used herein to mean, and is used interchangeably with, the term “and / or,” unless the context clearly indicates otherwise.

[0040] As used in a claim herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0041] Lipid PK / PD Modulators

[0042] One aspect of the invention provides a compound, or a pharmaceutically acceptable salt thereof, of Formula (I):wherein,R comprises an oligonucleotide-based agent;Li is a linking moiety;L2 is selected from the group consisting of: optionally substituted alkylene, optionally substituted arylene, 2-20 polyethylene glycol (PEG) units optionally interrupted by C(O)NRa, wherein R3 is selected from Ci-Ce alkyl and H, and a bond;Z is selected from the group consisting of: CH (wherein p is 1), CH2 (wherein p is 0), N (wherein p is 1). optionally substituted arylene. C(O)NRa (wherein p is 0), heterocyclene, and NR3 (wherein p is 0), wherein R3 is selected from Ci-Ce alkyl and H; p is 0 or 1, as valency permits;Yi and Y2 are each independently selected from the group consisting of: 2-20 polyethylene glycol (PEG) units, optionally substituted alkyl, optionally substituted cycloalkyl, and a bond;Xi and X2 are each independently selected from the group consisting of: C(O), C(O)NR2 and a bond, wherein R2 is Ci-Ce alky l or H; n and m are each independently an integer from 8 to 20;Wi and W2 are each independently selected from the group consisting of: H, COOH

[0043] In some embodiments Li is selected from the group consisting of -S-, maleimide,C(O)NH. and triazole. In further embodiments,some

[0044] In some embodiments, L2 is selected from the group consisting of:

[0045] In some embodiments, Z is selected from the group consisting of: O

[0046] In some embodiments, Y i is selected from the group consisting of:

[0047] In some embodiments, n is 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18, 19, or 20.

[0048] In one embodiment, the compound or pharmaceutically acceptable salt thereof, is of the formula:wherein R comprises an RNAi agent.

[0049] In one embodiment, the compound has the structure of LP161 :wherein R comprises an RNAi agent.

[0050] Another aspect of the invention provides for Lipid PK / PD modulator precursors and pharmaceutically acceptable salts thereof, which can be used to synthesize PK / PD modulators conjugated to oligonucleotide based agents.

[0051] In one embodiment, a lipid PK / PD modulator precursor or a pharmaceutically acceptable salt thereof, is of the formula:

[0052] In one embodiment, a lipid PK / PD modulator precursor, or a pharmaceutically acceptable salt thereof, has the structure:

[0053] Oligonucleotide-Based Agents, Including RJNAi Agents

[0054] As used herein, an '‘oligonucleotide-based agent” is a nucleotide sequence containing about 10-50 (e.g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14,10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40. 12 to 38, 12 to 36, 12 to 34,12 to 32. 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20. 12 to 18. 12 to 16. 12 to 14.14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32,14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48,16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34. 16 to 32, 16 to 30, 16 to 28,16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48. 18 to 46. 18 to 44, 18 to 42,18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22,18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34,20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44,22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30. 22 to 28, 22 to 26, 22 to 24,24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32,24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38,26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42,28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, to 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44. 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32. 32 to 50. 32 to 48. 32 to 46, 32 to44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40. 40 to 50, 40 to 48, 40 to 46, 40 to44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50. 44 to 48. 44 to 46, 46 to 50, 46 to48, or 48 to 50) nucleotides or nucleotide base pairs. In some embodiments, an oligonucleotide-based agent has a nucleobase sequence that is at least partially complementary to a coding sequence in an expressed target nucleic acid or target gene within a cell. In some embodiments, the oligonucleotide-based agents, upon delivery to a cell expressing a gene, are able to inhibit the expression of the underlying gene, and are referredto herein as “expression-inhibiting oligonucleotide-based agents.'’ The gene expression can be inhibited in vitro or in vivo.

[0055] “Oligonucleotide-based agents” include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, an oligonucleotide-based agent is a single-stranded oligonucleotide, such as an antisense oligonucleotide. In some embodiments, an oligonucleotide-based agent is a doublestranded oligonucleotide. In some embodiments, an oligonucleotide-based agent is a doublestranded oligonucleotide that is an RNAi agent.

[0056] In some embodiments, the oligonucleotide-based agent is / are an “RNAi agent,” which as defined herein is a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i. e. , inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0057] Typically, RNAi agents can be comprised of at least a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence. The length of an RNAi agent sense and antisense strands can each be 16 to 49 nucleotides in length. In some embodiments, the sense and antisense strands of an RNAi agent are independently 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands areindependently 21 to 24 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. The RNAi agents include an antisense strand sequence that is at least partially complementary to a sequence in the target gene, and upon delivery to a cell expressing the target, an RNAi agent may inhibit the expression of one or more target genes in vivo or in vitro.

[0058] Oligonucleotide-based agents generally, and RNAi agents specifically, may be comprised of modified nucleotides and / or one or more non-phosphodiester linkages. As used herein, a '‘modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides, non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids, 2',3'-seco nucleotide mimics (unlocked nucleobase analogues, locked nucleotides. 3'-O-methoxy (2' intemucleoside linked) nucleotides, 2 -F- Arabino nucleotides. 5'-Me, 2'-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides. 2'-modified nucleotides (i.e. a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides. 2'-deoxy nucleotides, 2'-methoxy ethyl (2'-O-2-methoxylethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides.

[0059] Moreover, one or more nucleotides of an oligonucleotide-based agent, such as an RNAi agent, may be linked by non-standard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). A modified intemucleoside linkage may be a non-phosphate-containing covalent intemucleoside linkage. Modified intemucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates. thiophosphates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters. alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl- phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates havinginverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0060] It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated in a single oligonucleotide- based agent or even in a single nucleotide thereof.

[0061] The RNAi agent sense strands and antisense strands may be synthesized and / or modified by methods known in the art. Additional disclosures related to RNAi agents may be found, for example, in the disclosure of modifications may be found, for example, in International Patent Application No. PCT / US2017 / 045446 (WO2018027106) to Arrowhead Pharmaceuticals, Inc., which also is incorporated by reference herein in its entirety.

[0062] Modified Nucleotides

[0063] In some embodiments, an RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab), 2'-modified nucleotides, 3' to 3’ linkages (inverted) nucleotides (represented herein as invdN, invN, invn), modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3 '-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as Ni / x \ or NUNA), locked nucleotides (represented herein as NLNA or NLNA), 3'-O-methoxy (2' intemucleoside linked) nucleotides (represented herein as 3'-0Men), 2’-F-Arabino nucleotides (represented herein as NfANA or MANA), 5’-Me, 2'- fluoro nucleotide (represented herein as 5Me-Nf). morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides (cPrpN). 2'-modified nucleotides (i.e., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (represented herein as a lower case letter ‘n’ in a nucleotide sequence). 2'-deoxy-2'-fluoro nucleotides (also referred to herein as 2'-fluoro nucleotide, and represented herein as Nf), 2'- deoxy nucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides (also referred to herein as 2'-M0E, and represented herein as NM), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can beincorporated in a single target RNAi agent or even in a single nucleotide thereof. The target RNAi agent sense strands and antisense strands can be synthesized and / or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.

[0064] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine). 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, inosine, xanthine, hypoxanthine. 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2 -isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2 -thiothymine, 2 -thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7 -methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine. 7-deazaadenine, 3 -deazaguanine, and 3 -deazaadenine.

[0065] In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RN Ai agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e.. 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e.. 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. As used herein, an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is an unmodified ribonucleotide.

[0066] Modified Internucleoside Linkages

[0067] In some embodiments, one or more nucleotides of an RNAi agent are linked by nonstandard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). Modified intemucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower case “s"’), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters. alkyl phosphonates (e.g., methyl phosphonates or 3 '-alkylene phosphonates), chiral phosphonates,phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates). thionoalkyl-phosphonates. thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter- sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0068] In some embodiments, a sense strand of an RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of an RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of an RNAi agent can contain 1, 2, 3. or 4 phosphorothioate linkages, an antisense strand of an RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.

[0069] In some embodiments, an RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate intemucleoside linkage is at the 5 ’ end of the sense strand, and another phosphorothioate linkage is at the 3 ’ end of the sense strand. In some embodiments, two phosphorothioate intemucleoside linkage are located at the 5’ end of the sense strand, and another phosphorothioate linkage is at the 3’ end of the sense strand. In some embodiments, the sense strand does not include any phosphorothioate intemucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5’ and 3’ endsand the optionally present inverted abasic residue terminal caps. In some embodiments, the targeting ligand is linked to the sense strand via a phosphorothioate linkage.

[0070] In some embodiments, an RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, three phosphorothioate intemucleoside linkages are located between positions 1-4 from the 5’ end of the antisense strand, and a fourth phosphorothioate intemucleoside linkage is located between positions 20-21 from the 5’ end of the antisense strand. In some embodiments, an RNAi agent contains at least three or four phosphorothioate intemucleoside linkages in the antisense strand.

[0071] In some embodiments, an RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2’-modified nucleoside is combined with modified intemucleoside linkage.

[0072] Targeting Ligands and Targeting Groups

[0073] In some embodiments, ohgonucleotide-based agents may also be conjugated to a targeting ligand or targeting group to form a compound according to the instant invention. Targeting ligands or targeting groups enhance the pharmacokinetic or biodistribution properties of a conjugate or RNAi agent to which they are attached to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the conjugate or RNAi agent. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency for the target to which it is directed. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules. In some embodiments, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and / or ribitol (abasic ribose) residues, which in some instances can serve as linkers. In some embodiments, a targeting group comprises one or more glucagon-like peptide 1 receptor (GLP1R) targeting ligands.

[0074] In some embodiments, a targeting ligand enhances the ability of the RNAi agent to bind to a particular cell receptor on a cell of interest. In some embodiments, the targeting ligands conjugated to RNAi agents described herein have affinity for GLP1R.

[0075] In some embodiments, the RNAi agents described herein are conjugated to targeting groups. Targeting groups comprise two or more targeting ligands.

[0076] In some embodiments, an RNAi agent disclosed herein is linked to one or more

[0077] In some embodiments, targeting ligands are conjugated to an RNAi agent using a “click” chemistry reaction. In some embodiments, RNAi agents are functionalized with one or more alkyne-containing groups, and targeting ligands include azide-containing groups. Upon reaction, azides and alkynes form triazoles. An example reaction scheme is shown below:wherein TL comprises a targeting ligand, and Rzzz comprises an RNAi agent.

[0078] RNAi agents may comprise more than one targeting ligand. In some embodiments, RNAi agents comprise 1-20 targeting ligands. In some embodiments, RNAi agents comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, or 19 targeting ligands to 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19 or 20 targeting ligands.

[0079] In some embodiments. RNAi agents described herein comprise a targeting group, which includes 2 or more targeting ligands. In some embodiments, a targeting group may be conjugated at the 5’ or 3’ end of the sense strand of an RNAi agent. In some embodiments, a targeting group may be conjugated to an internal nucleotide on an RNAi agent. In some embodiments, a targeting group may consist of two targeting ligands linked together, referred to as a “bidentate” targeting group. In some embodiments, a targeting group may consist of three targeting ligands linked together, referred to as a “tridentate” targeting group. In some embodiments, a targeting group may consist of four targeting ligands linked together, referred to as a “tetradentate” targeting group.

[0080] In some embodiments, RNAi agents may comprise both a targeting group conjugated to the 3’ or 5’ end of the sense strand, and additionally targeting ligands conjugated tointernal nucleotides. In some embodiments a tridentate targeting group is conjugated to the 5’ end of the sense strand of an RNAi agent, and at least one targeting ligand is conjugated to an internal nucleotide of the sense strand. In further embodiments, a tridentate targeting group is conjugated to the 5’ end of the sense strand of an RNAi agent, and four targeting ligands are conjugated to internal nucleotides of the sense strand.

[0081] Linking Groups and Delivery Agents

[0082] In some embodiments, the oligonucleotide-based agent, such as RNAi agents described herein, contains or is conjugated to one or more non-nucleotide groups including, but not limited to a linking group or a delivery agent. The non-nucleotide group can enhance targeting, delivery, or attachment of the RNAi agent. Examples of linking groups are provided in Table 1. The non-nucleotide group can be covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, an RNAi agent contains a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of an RNAi agent sense strand. A non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0083] In some embodiments, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.

[0084] The RNAi agents described herein can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine), at the 5'-terminus and / or the 3'- terminus. The reactive group can be used subsequently to attach a targeting moiety using methods typical in the art.

[0085] For example, in some embodiments, the RNAi agents disclosed herein are synthesized having an NH2-C6 group at the 5'-terminus of the sense strand of the RNAi agent. The terminal amino group subsequently can be reacted to form a conjugate with, for example, a group that includes a compound having affinity for GLP1R (i.e., a GLP1R targeting ligand) or a PK enhancer. In some embodiments, the RNAi agents disclosed herein are synthesized having one or more alkyne groups at the 5’-terminus of the sense strand of the RNAi agent. The terminal alkyne group(s) can subsequently be reacted to form a conjugate with, for example, a group that includes a targeting ligand.

[0086] In some embodiments, a targeting group comprises a GLP1R targeting ligand. In some embodiments, a GLP1R targeting ligand includes a compound that has affinity to GLP1R. The use of GLP1R targeting ligands can facilitate cell-specific targeting to cells having the respective GLP1 receptors on its respective surface, and binding of the GLP1R targeting ligand can facilitate entry of the RNAi agent, to which it is linked, into cells such as cardiomyocytes. Targeting ligands, targeting groups, and / or PK / PD modulators can be attached to the 3' and / or 5' end of the RNAi agent, and / or to internal nucleotides on the RNAi agent, using methods generally known in the art. The preparation of targeting ligand and targeting groups, such as GLP1R ligands, is described in Example 3 below.

[0087] Some embodiments of the present disclosure include pharmaceutical compositions for delivering an RNAi agent to a cardiomyocyte in vivo. Such pharmaceutical compositions can include, for example, an RNAi agent conjugated to a targeting group that comprises a GLP1R targeting ligand that has affinity for GLP1R. In some embodiments, the targeting ligand is comprised of a compound having affinity7for GLP1R.

[0088] In some embodiments, the RNAi agent is synthesized having present a linking group, which can then facilitate covalent linkage of the RNAi agent to a targeting ligand, a targeting group, a PK / PD modulator, or another type of delivery agent. The linking group can be linked to the 3' and / or the 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5' end of an RNAi agent sense strand. Examples of linking groups, include, but are not limited to: C6-SS-C6, reactive groups such a primary amines and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, trialkyne functionalized groups, ribitol, and / or PEG groups.

[0089] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting ligand, targeting group, PK / PD modulator, or delivery agent) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and / or length to the linkage. Spacers include, but are not limited to, alky l groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.

[0090] In some embodiments, targeting groups are linked to the RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed to have a linker readily present to facilitate the linkage to an RNAi agent. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents can be linked to their respective targeting groups using the same linkers. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0091] RNAi agents whether modified or unmodified, may contain 3' and / or 5' targeting group(s), linking group(s), and / or may be conjugated with, or comprise, PK / PD modulator(s). Any of the RNAi agent sequences listed in Table 2, or are otherwise described herein, which contain a 3' or 5' targeting ligand, targeting group, PK / PD modulator, or linking group, can alternatively contain no 3' or 5' targeting ligand, targeting group, linking group, or PK / PD modulator, or can contain a different 3' or 5' targeting ligand, targeting group, linking group, or PK / PD modulator including, but not limited to, those depicted in Table 1. Any of the RNAi agent duplexes listed in Table 2 whether modified or unmodified, can further comprise a targeting ligand, targeting group, linking group, or PK / PD modulator, and the targeting group or linking group can be attached to the 3' or 5' terminus of either the sense strand or the antisense strand of the RNAi agent duplex.

[0092] In some embodiments, a linking group may be conjugated synthetically to the 5’ or 3’ end of the sense strand of an RNAi agent described herein. In some embodiments, a linking group is conjugated synthetically to the 5’ end of the sense strand of an RNAi agent. In some embodiments, a linking group conjugated to an RNAi agent may be a trialkyne linking group.

[0093] Examples of certain PK / PD modulators, targeting ligands, modified nucleotides, and linking groups, are provided in Table 1 .Table 1: Structures Representing Various Modified Nucleotides and Linking Groups.

[0094] Alternatively, other linking groups known in the art may be used.

[0095] In addition or alternatively to linking an RNAi agent to one or more targeting ligands, targeting groups, and / or PK / PD modulators, in some embodiments, a delivery agent may be used to deliver an RNAi agent to a cell or tissue. A delivery7agent is a compound that can improve delivery of the RNAi agent to a cell or tissue, and can include, or consist of. but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.

[0096] In some embodiments, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169, and WO 2012 / 083185. WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference), or other delivery systems available in the art.

[0097] Pharmaceutical Compositions

[0098] In some embodiments, the present disclosure provides pharmaceutical compositions that include, consist of, or consist essentially of. one or more compounds of Formula (I).

[0099] As used herein, a ‘"pharmaceutical composition” comprises a pharmacologically effective amount of an Active Pharmaceutical Ingredient (API), and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid inprocessing of the drug deliver}' system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0100] Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.

[0101] The pharmaceutical compositions described herein can contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically-active material. Pharmaceutically-active materials include, but are not limited to: anti-pruritics, astringents, local anesthetics, or antiinflammatory agents (e.g.. antihistamine, diphenhydramine, etc.), small molecule drug, antibody, antibody fragment, aptamers, and / or vaccines.

[0102] The pharmaceutical compositions may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other agents with a known therapeutic benefit.

[0103] The pharmaceutical compositions can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.Administration can be made by any way commonly known in the art. such as, but not limited to, topical (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal), epidermal, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal (e.g., via an implanted device), intracranial, intraparenchymal. intrathecal, and intraventricular, administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous inj ection. The pharmaceutical compositions may be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally or percutaneously.for example using ointments, creams, gels, or solutions; or parenterally, for example using injectable solutions.

[0104] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0105] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0106] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of any of the ligands described herein that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present any of the ligands described herein for both intra-articular and ophthalmic administration.

[0107] The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymerscan be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0108] A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e g., antihistamine, diphenhydramine, etc.). As used herein, “pharmacologically effective amount,’' “therapeutically effective amount,” or simply “effective amount” refers to that amount of an the pharmaceutically active agent to produce a pharmacological, therapeutic or preventive result.

[0109] Medicaments containing compounds of Formula (I) are also an object of the present invention, as are processes for the manufacture of such medicaments, which processes comprise bringing one or more compound of Formula (I), and, if desired, one or more other substances with a known therapeutic benefit, into a pharmaceutically acceptable form.

[0110] The described compounds of Formula (I) and pharmaceutical compositions comprising compounds of Formula (I) disclosed herein may be packaged or included in a kit, container, pack, or dispenser. The compounds of Formula (I) and pharmaceutical compositions comprising the compounds of Formula (I) may be packaged in pre-filled syringes or vials.

[0111] Methods of Treatment and Inhibition of Expression

[0112] The compounds of Formula (I) disclosed herein can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of such compounds. In some embodiments, the compounds of Formula (I) disclosed herein can be used to treat a subject (e.g., a human) that would benefit from reduction and / or inhibition in expression of a target mRNA and / or protein levels, for example, a subject that has been diagnosed with or is suffering from symptoms related to pulmonary hypertension.

[0113] In some embodiments, the subject is administered a therapeutically effective amount of one or more compounds of Formula (I) disclosed herein. Treatment of a subject can include therapeutic and / or prophylactic treatment. The subject is administered a therapeutically effective amount of one or more compounds of Formula (I) described herein. The subject can be a human, patient, or human patient. The subject may be an adult.adolescent, child, or infant. Administration of a pharmaceutical composition described herein can be to a human being or animal.

[0114] The compounds of Formula (I) described herein can be used to treat at least one symptom in a subject having a disease or disorder related to a target gene, or having a disease or disorder that is mediated at least in part by the expression of the target gene. In some embodiments, the compounds of Formula (I) are used to treat or manage a clinical presentation of a subject with a disease or disorder that would benefit from or be mediated at least in party by a reduction in mRNA of a target gene. The subject is administered a therapeutically effective amount of one or more of the compounds of Formula (I) or compositions described herein. In some embodiments, the methods disclosed herein comprise administering a composition comprising a compound of Formula (I) described herein to a subject to be treated. In some embodiments, the subject is administered a prophy tactically effective amount of any one or more of the described compounds of Formula (I), thereby treating the subject by preventing or inhibiting the at least one symptom.

[0115] In certain embodiments, the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by target gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the compounds of Formula (I) described herein.

[0116] In some embodiments, the gene expression level and / or mRNA level of a target gene in a subject to whom a compound of Formula (I) described herein is administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the compound or to a subject not receiving the compound. The gene expression level and / or mRNA level in the subject may be reduced in a cell, group of cells, and / or tissue of the subject.

[0117] In some embodiments, the target protein level in a subject to whom a compound of Formula (I) described herein has been administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. 80%. 85%. 90%. 95%. 96%. 97%. 98%. 99%. or greater than 99% relative to the subject prior to being administered the compound or to a subject not receiving the compound. The protein level in the subject may be reduced in a cell, group of cells, tissue, blood, and / or other fluid of the subject.

[0118] A reduction in target mRNA levels and / or target protein levels can be assessed by any methods known in the art. As used herein, a reduction or decrease in target mRNA leveland / or protein level are collectively referred to herein as a reduction or decrease in target gene and / or protein levels or inhibiting or reducing the expression of a target gene.

[0119] In some embodiments, compounds of Formula (I) described herein may be used in the preparation of a pharmaceutical composition for use in the treatment of a disease, disorder, or symptom that is mediated at least in part by target gene expression. In some embodiments, the disease, disorder, or symptom that is mediated at least in part by target gene expression is pulmonary hypertension.

[0120] In some embodiments, methods of treating a subject are dependent on the body weight of the subject. In some embodiments, compounds of Formula (I) may be administered at a dose of about 0.05 mg / kg to about 40.0 mg / kg of body weight of the subject. In other embodiments compounds of Formula (I) may be administered at a dose of about 5 mg / kg to about 20 mg / kg of body weight of the subject.

[0121] In some embodiments, compounds of Formula (I) may be administered in a split dose, meaning that two doses are given to a subject in a short (for example, less than 24 hour) time period. In some embodiments, about half of the desired daily amount is administered in an initial administration, and the remaining about half of the desired daily amount is administered approximately four hours after the initial administration.

[0122] In some embodiments, compounds of Formula (I) described herein may be administered once a week (i.e., weekly). In other embodiments, compounds of Formula (I) described herein may be administered biweekly (once every other week).

[0123] In some embodiments, compounds of Formula (I) described herein or compositions containing such compounds may be used for the treatment of a disease, disorder, or symptom that is mediated at least in part by target gene expression. In some embodiments, the disease, disorder or symptom that is mediated at least in part by target gene expression is pulmonary hypertension.

[0124] Another aspect of the invention provides for a method of reducing a target gene expression in vivo, the method comprising introducing to a cell a compound of Formula (I), described herein, wherein the compound comprises an RNAi agent at least substantially complementary to the target gene. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell is within a subject. In some embodiments, the subject has been diagnosed with a disease or disorder that is treated, prevented or ameliorated by reducing expression of the target gene.

[0125] Another aspect of the invention provides for the use of any one of the lipid PK / PD modulators conjugated to an oligonucleotide-based agent described herein for the treatment,prevention, or amelioration of a disease or disorder. In some embodiments, the disease or disorder is pulmonary hypertension.

[0126] Cells, Tissues, and Non-Human Organisms

[0127] Cells, tissues, and non-human organisms that include at least one of the compounds of Formula (I), described herein is contemplated. The cell, tissue, or non-human organism is made by delivering the compound of Formula (I) to the cell, tissue, or non-human organism by any means available in the art. In some embodiments, the cell is a mammalian cell, including, but not limited to, a human cell. In some embodiments the cell is a cardiomyocyte.

[0128] The above provided embodiments and items are now illustrated with the following, non-limiting examples.OTHER EMBODIMENTS

[0129] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.Embodiment 1. A compound of Formula (I):wherein,R comprises an oligonucleotide-based agent;Li is a linking moiety;L2 is selected from the group consisting of: optionally substituted alkylene, optionally substituted arylene, 2-20 polyethylene glycol (PEG) units optionally interrupted by C(O)NRs, wherein Rs is selected from Ci-Ce alkyd and H, and a bond;Z is selected from the group consisting of: CH (wherein p is 1), CH2 (wherein p is 0), N (wherein p is 1). optionally substituted arylene. C(O)NRs (wherein p is 0), heterocyclene, and NRs (wherein p is 0), wherein Rs is selected from Ci-Ce alkyl and H; p is 0 or 1, as valency permits;Yi and Y2 are each independently selected from the group consisting of: 2-20 polyethylene glycol (PEG) units, optionally substituted alkyl, optionally substituted cycloalkyl, and a bond;Xi and X2 are each independently selected from the group consisting of: C(O), C(O)NR2 and a bond, wherein R2 is Ci-Ce alky l or H; n and m are each independently an integer from 8 to 20;Wi and W2 are each independently selected from the group consisting of: H, COOHEmbodiment 2. The compound of embodiment 1. wherein Li is selected from the group consisting of -S-, maleimide, C(O)NH, and triazole,, and -C(O)-.Embodiment 3. The compound of embodiment 1 or embodiment 2, wherein Li isEmbodiment 4. The compound of embodiment 1 or embodiment 2, wherein Li isEmbodiment 5. The compound of any one of embodiments 1-4, wherein L2 is selectedEmbodiment 6. The compound of any one of embodiments 1-5, wherein Z is selected from the group consisting of:Embodiment 7. The compound of any one of embodiments 1-6, wherein Yi is selected from the group consisting of :Embodiment 8. The compound of any one of embodiments 1-5, wherein Xi isC(O)NR2.Embodiment 9. The compound of embodiment 6. wherein R2 is H.Embodiment 10. The compound of any one of embodiments 1-7, wherein n is 13. 14.15, or 16.Embodiment 11. The compound of any one of embodiments 1-8, wherein p is 0.Embodiment 12. The compound of any one of embodiments 1-10, w herein p is 1.Embodiment 13. The compound of embodiment 12, wherein Z is N.Embodiment 14. The compound of any one of embodiments 1-13, wherein Wi is H.Embodiment 15. The compound of any one of embodiments 1-14, wherein R is anRNAi agent.Embodiment 16. A compound, or a pharmaceutically acceptable salt thereof, selectedEmbodiment 17. A compound, or a pharmaceutically acceptable salt thereof, of the formula:wherein R is an RNAi agent.Embodiment 18. A compound having of the structure:Embodiment 20. A method of delivering an RNAi agent to a cardiomyocyte, comprising administering to a subject the compound of any one of embodiments 1-17.Embodiment 21. A method of inhibiting the expression of a gene expressed in a cardiomyocyte, comprising administering to a subject the compound of any one of embodiments 1-17.Embodiment 22. The compound of any one of embodiments 1-17, for use in inhibiting expression of a gene expressed in a cardiomyocyte.Embodiment 23. A method of making a compound of Formula I:wherein,R comprises an oligonucleotide-based agent;Li is a linking moiety;L2 is selected from the group consisting of: optionally substituted alkylene, optionally substituted arylene, 2-20 polyethylene glycol (PEG) units optionally interrupted by C(O)NR3, wherein Rs is selected from Ci-Ce alkyl and H, and a bond;Z is selected from the group consisting of: CH (wherein p is 1), CH2 (wherein p is 0), N (wherein p is 1). optionally substituted arylene. C(O)NRs (wherein p is 0), heterocyclene, and NRs (wherein p is 0), wherein Rs is selected from Ci-Ce alkyl and H; p is 0 or 1, as valency permits;Yi and Y2 are each independently selected from the group consisting of: 2-20 polyethylene glycol (PEG) units, optionally substituted alkyl, optionally substituted cycloalkyl, and a bond;Xi and X2 are each independently selected from the group consisting of: C(O), C(O)NR2 and a bond, wherein R2 is Ci-Ce alkyl or H; n and m are each independently an integer from 8 to 20;Wi and W2 are each independently selected from the group consisting of: H, COOH andcomprising reacting a compound of embodiment 19 with an RNAi agent comprising a reactive group to form the compound of Formula I.Embodiment 24. The method of embodiment 23, wherein the reactive group is selected from the group consisting of: disulfide, amine, and alkyne.Embodiment 25. The method of embodiment 23, wherein the compound reacted with an RNAi agent comprising a reactive group comprises the formula:LP161-p.EXAMPLES

[0130] The following examples are not limiting and are intended to illustrate certain embodiments disclosed herein.

[0131] Unless expressly stated otherwise, numerals used to refer to compounds of a given example are only made with reference to that particular example and may not refer to any other examples disclosed herein. Compounds that are disclosed in various tables throughout the detailed description (e.g., LP161-a, LP161-p, LP161) are referred to consistently throughout the examples herein.Example 1. Syntheses of RNAi agents and Compositions.

[0132] The following describes the general procedures for the syntheses of certain RNAi agents, and conjugates thereof, that are illustrated in the non-limiting Examples set forth herein.

[0133] Synthesis of RNAi Agents . RNAi agents can be synthesized using methods generally known in the art. For the synthesis of the RNAi agents illustrated in the Examples set forth herein, the sense and antisense strands of the RNAi agents were synthesized according to solid phase phosphoramidite technology used in oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an Oligopilot 100 (GE Healthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 A or 600A, obtained from Prime Synthesis, Aston, PA,USA) or polystyrene (obtained from Kinovate, Oceanside, CA, USA). All RNA and 2'- modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA), or Hongene Biotech (Morrisville, NC, USA). Specifically, the following 2'-O-methyl phosphoramidites that were used include the following: ('5'-O-dimetho.\ytntyl-N6-(benzoyl)-2'-O-mcthyl-adenosinc-3'-O-('2-cyanoethyl-N.N- diisopropylamino) phosphoramidite. 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-O-methyl-cytidine- 3'-O-(2-cyanoethyl-N.N-diisopropyl-amino) phosphoramidite, (5'-O-dimethoxytrityl-N2- (isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5 '-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N- diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidites and 2'-O- propargyl phosphoramidites carried the same protecting groups as the 2'-O-methyl phosphoramidites. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N- diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia). The inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N- diisopropylamino) phosphoramidites were purchased from ChemGenes. The following UNA phosphoramidites that were used included the following: 5'-(4,4’-Dimethoxytrityl)-N6- (benzoyl)-2', 3 '-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 5'-(4,4'-Dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2- cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidite, 5'-(4,4'-Dimethoxytrityl)-N-isobutyryl- 2', 3 '-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-Dimethoxy-trityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N- di-iso- propyl)] -phosphoramidite. In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl l,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile or a 200mM solution of xanthane hydride (TCI America, Portland, OR, USA) in pyridine was employed.

[0134] TFA aminolink phosphoramidites were also commercially purchased (ThermoFisher) to introduce the (NH2-C6) reactive group linkers. TFA aminolink phosphoramidite w as dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 ) were added. 5- Benzylthio-lH-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-lH-tetrazole (ETT. 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 90 sec (2' O-Me), and 60 sec (2' F). Trialkyne-containing phosphoramidites were synthesized to introduce the respective (TriAik#) linkers. When used in connection with the RNAi agents presented in certain Examples herein, trialkyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amiditeswere dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 ) were added. 5- Benzylthio-lH-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-lH-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 90 sec (2' O-Me), and 60 sec (2' F).

[0135] For some RNAi agents, a linker, such as a C6-SS-C6 or a 6-SS-6 group, was introduced at the 3’ terminal end of the sense strand. Pre-loaded resin was commercially acquired with the respective linker. Alternatively, for some sense strands, a dT resin was used and the respectively linker was then added via standard phosphoramidite synthesis.

[0136] C 'leavage and deprotection of support bound oligomer. After finalization of the solid phase synthesis, the dried solid support was treated with a 1: 1 volume solution of 40 weight (wt.) % methylamine in water and 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30 °C. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0137] Purification. Crude oligomers were purified by anionic exchange HPLC using a TSKgel® SuperQ-5PW Bpm column (available from Tosoh Biosciences) and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA. pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex® G25 fine (available from Sigman Aldrich)with a running buffer of lOOmM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile or filtered water.

[0138] Annealing. Complementary7strands were mixed by combining equimolar RNA solutions (sense and antisense) in l x PBS (Phosphate-Buffered Saline, l x, Coming, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25 °C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 x PBS. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the duplex concentration. The conversion factor used was either 0.037 mg / (mL-cm) or was calculated from an experimentally determined extinction coefficient.Example 2. Synthesis of Linking Groups

[0139] Synthesis of L4

[0140] To a solution of compound 1 (3.00 g) in DMF was added CS2CO3 (7.71 g) at room temperature. Compound 2 (1 .85 mL) was then added slowly. The resulting reaction mixture was stirred overnight under N2 (g). Approximately full conversion to desired product by LC- MS was then confirmed. The reaction mixture was quenched with NaHCCL (10 mL). The product was extracted with EtOAc (5 x 10 mL) and then washed with water (3 x 8 mL) and brine (8 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary' phase with a gradient of hexanes to EtOAc (0-30%), in which the product eluted at 14% B. Compound 3 was concentrated under vacuum to provide a white solid. LC-MS: calculated [M+H]+ 191.06 m / z. observed 191.23 m / z.

[0141] To a solution of compound 3 (2.87 g) in 1 :1 THF / water was added LiOH (1.08 g) at room temperature under normal atmosphere. The reaction mixture was stirred until full conversion of compound 3 was observed by LC-MS. Residual starting material was extracted via EtOAc, and then aqueous phase was acidified with 6 N HC1 to a pH of approximately 3. Compound 4 crashed out as a white solid and was filtered over vacuum and washed with water. Due to its wet / sticky nature, solvent was required to transfer the solid to a round bottom flask; material was transferred via MeOH and DCM. Due to poor solvation in either solvent and the combination, the material could not to be dried over Na2SC>4. Compound 4 was concentrated under vacuum to provide a white, fluffy cry stalline solid and was used directly without further purification. LC-MS: calculated [M+H]+ 177.05 m / z, observed 177.19 m / z.

[0142] To a solution of compounds 4 (1.00 g) and 5 (1.04 g) in DMF (10.0 mL) under N2(g) was added EDC hydrochloride salt (1.20 g) at room temperature. The reaction mixture was allowed to stir until full conversion was observed by LC-MS. Due to an inability to successfully observe the product after overnight stirring, the reaction mixture was quenched with NaHCOs. The resulting precipitate was confirmed to contain starting materials via LC- MS and was filtered over vacuum, attempted to be re-suspended in MeOH / DCM, and then concentrated under vacuum. The mixture was then re-solvated in DMF, dried over Na2SO4. filtered over vacuum, and rinsed with DMF. EDC was re-added to the filtrate (i.e., compounds 4 and 5) in DMF, and the resultant mixture was allowed to stir overnight at room temperature. The reaction mixture was directly concentrated and azeotroped with MeOH and PhMe for isolation. The residue was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 0-20% MeOH in DCM. L4 eluted at 0% B to provide a white solid. LC- MS: calculated [M+H]+ 325.04 m / z, observed 325.35 m / z.Example 3. Synthesis of GLP1R Targeting Ligands

[0143] The following general procedures were used as indicated throughout the examples provided herein.

[0144] General Procedure I: Amide Coupling

[0145] To a round bottom flask containing carboxylic acid (1 eq) in DCM (0.2 M) was added COMU (1.2 eq) and DIPEA (2.5 eq). The mixture was stirred at room temperature for 30 min. Then, amine (1 eq) was added, and the mixture was continued to stir for 2 hours. After, the reaction mixture was concentrated under reduced pressure, loaded onto celite, and purified using CombiFlash chromatography.

[0146] General Procedure II: Alkylation of Phenols

[0147] To a round bottom flask containing phenol derivative (1 eq) in ACN (0.2 M) was added K2CO3 (3 eq) and corresponding alkyl bromide (1.2 eq). The reaction was stirred at 85°C for 12 hours. After, the reaction was cooled to room temperature, diluted with water (5 mL) and extracted thrice with ethyl acetate (10 mL). The combined organics were dried over Na2SO4, concentrated under reduced pressure, and purified via CombiFlash chromatography.

[0148] General Procedure III: Acylation

[0149] To a round bottom flask containing amine (1 eq) in DCM or THF (0.2 M) was added TEA (2 eq) and corresponding acid chloride, activated ester, or isocyanate (1.2 eq). The mixture was stirred at room temperature. Once LCMS indicated complete reaction, themixture was concentrated under reduced pressure, loaded onto celite, and purified via CombiFlash chromatography.

[0150] General Procedure IV: Oxidation of Methylthiol to Sulfone

[0151] To a round bottom flask containing methylthiol intermediate (1 eq) in anhydrous DCM (0.2 M) was cooled to 0°C and mCPBA (6 eq) was added. The solution was warmed to room temperature and stirred overnight. Once the reaction was complete, the mixture was quenched with sat. aqueous Na2S20s solution (10 mL) and continued to stir for 30 min. Then, the mixture was poured in H2O (15 mL) and extracted thrice with DCM (3 x 20 mL). The combined organics were dried over Na2SOr and concentrated under reduced pressure. The crude was dissolved in ethyl acetate (25 mL) and washed twice with NaHCOs (2 x 20 mL), twice with H2O (2 x 20 mL). and once with brine (20 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was purified via CombiFlash chromatography.

[0152] General Procedure V: Acidic Deprotection of MOM ethers, Tertbutyl Esters, and Boc-Containing Amines

[0153] To a round bottom flask containing MOM ether, tertbutyl ester, or Boc-protected amine was added a mixture of TFA: DCM (3: 1). The reaction was stirred for 1 hour and then neutralized with saturated NaHCOs solution. The neutralized solution was then extracted thrice with ethyl acetate (10 mL). The combined organics were dried over Na2SO4. concentrated under reduced pressure, and purified via CombiFlash chromatography.

[0154] General Procedure VI: Methyl Ester Hydrolysis with LiOH

[0155] To a round bottom flask containing methyl ester (1 eq) in MeOH: H2O (2: 1) was added LiOH (10 eq). The mixture was stirred at room temperature for 12 hours. After, the mixture was concentrated under reduced pressure, diluted with water (5 mL) and acidified to pH = 2 with 6 M HC1 to give an off-white precipitate which was collected via vacuum filtration.

[0156] General Procedure VII: Fmoc Deprotection

[0157] To a round bottom flask containing Fmoc protected amine (1 eq) was added a solution of 25% piperidine in DMF (5 mL). The reaction was stirred for 1 hour at room temperature. After, the solution was diluted with H2O (10 mL) and extracted thrice with ethyl acetate (3 x 20 mL). The combined organics were w ashed once with H2O (10 mL), once with brine (10 mL), dried over Na2SC>4, and concentrated under reduced pressure. The crude was purified via CombiFlash chromatography.

[0158] General Procedure VIII: SNAR reaction of a 3-fluoro-4-nitrobenzoate

[0159] To a flame dried flask, 3-fluoro-4-nitrobenzoate (1 eq) and primary amine (1 eq) were dissolved in solvent (0.2 M). Base was added, and the mixture was stirred for 3-16 hours at the indicated temperature. The volatiles were removed under reduced pressure, and the crude material was purified via flash chromatography on silica gel to yield the desired 3-amino-4- nitrobenzoate.

[0160] General Procedure IX; Nitro reduction of a 3-amino-4-nitrobenzoate

[0161] To a flame dried flask was added 3-amino-4-nitrobenzoate (1 eq) and 10% palladium on carbon (10% w / w) in a mixture of 3: 1 THF / MeOH (0.2 M). Then, hydrogen gas was bubbled into the suspension for 15 minutes. The reaction continued to stir under an H2 atmosphere for 2 hours. The solids were filtered off using a celite plug, which was washed with a 1: 1 mixture of MeOH / THF three times. The volatiles were removed to afford the desired 4-amino-3-(methylamino)benzoate.

[0162] General Procedure X; 2-(Chloromethyl)-benzo[d]imidazole-6-carboxylate synthesis

[0163] A 4-amino-3-(methylamino)benzoate (1 eq) was dissolved in MeCN (0.2 M). 2- chloro- 1,1,1 -trimethoxy ethane (3 eq) and p-toluenesulfonic acid monohydrate (pTSA FbO) (0.5 eq) were added, and the reaction was heated to 40-60°C for 2 hours. The volatiles w ere removed under reduced pressure, and the crude material w as purified via flash chromatography on silica gel to afford the desired 2-(chloromethyl)-benzo[d]imidazole-6- carboxylate.

[0164] General Procedure XI; Alkylation of a 3-amino-4-nitrobenzoate

[0165] A 2-(chloromethyl)-benzo[d]imidazole-6-carboxylate (1 eq) and a secondary' amine (1.2 eq) were dissolved in MeCN (0.2 M), then K2CO3 (5 eq) was added and the reaction heated to 50°C for 3-16 hours. Volatiles were removed under reduced pressure and the crude material was purified via flash chromatography on silica gel to afford the desired ester product.

[0166] General Procedure XII; Ester deprotection and amidation

[0167] To a solution of ester in MeCN (0.2 M) was added a 1 M aqueous solution of 1,5,7- triazabicyclo[4.4.0]-dec-5-ene. The solution stirred for 3-16 hours. The pH of the solution was adjusted to 4 using 1 M HC1, then washed three times with ethyl acetate. The organic layers were combined and dried over sodium sulfate. The volatiles were removed, then the crude material was dissolved in methylene chloride (0. 1 M). COMU (2 eq) and DIEA (3 eq) were added, and the reaction stirred for 15 minutes, followed by addition of azido-PEG4- amine. The reaction was stirred for 2 hours, then the volatiles w ere removed under reducedpressure and the crude material purified via flash chromatography on silica gel to afford the desired GLP1R-SM product.Scheme 113 GLP1 R-SM4-P

[0168] Reagents and Conditions: (a) COMU, DIPEA, DMF, rt, 2 hr, 67%. (b) TFA: DCM (3: 1). rt, 1 h, 95%, (c) K2CO3, ACN. 85°C, 12 hr, 71%

[0169] Synthesis of 12:

[0170] 12 was synthesized following General Procedure I. The product was purified using hexanes: ethyl acetate (80:20) to give an oil in 67% yield. LC-MS [M+H]+478.0766 m / z, observed 478.0764 m / z.

[0171] Synthesis of 13:

[0172] 13 was synthesized starting from 12 following General Procedure V to give product as an off-white solid in 95% yield. LC-MS [M+H]+ 434.0504 m / z, observed 434.0501 m / z.

[0173] Synthesis of GLPlR-SM4-p:

[0174] GLP1R-SM4 was synthesized from 12 following General Procedure II. The product was purified using DCM: MeOH (98:2) to give an oil in 71% yield. LC-MS [M+H]+ 679.1880 m / z, observed 679.1878 m / z.Scheme 2. Synthesis of GLPlR-SM19-p

[0175] Reagents and Conditions: (a) K2CO3, ACN, 85°C, 12 hr, 68%. (b) TFA: DCM (3: 1), rt, 1 h, 95%. (c) NHS, EDC HC1, DCM. 30°C, 12 h. 73%.

[0176] Synthesis of E3:

[0177] E3 was synthesized according to General Procedure II to give product as an oil in 68% yield. LC-MS [M+H]+ 738.2390 m / z, observed 738.2392 m / z.

[0178] Synthesis of 18:

[0179] 18 was synthesized from E3 following General Procedure V to give product as an off- white solid in 95% yield. LC-MS [M+H]+ 550.0978 m / z, observed 550.0977 m / z.

[0180] Synthesis of GLPlR-SM19-p:

[0181] To a solution of 18 (1 eq) and NHS (1.1 eq) in DCM (0.2 M) was added EDC HC1 (2 eq). The mixture was stirred at 30°C for 12 hours. Then, the reaction was concentrated under reduced pressure and purified via CombiFlash column chromatography using DCM: MeOH (98:2) to give produce as an off-white solid in 73% yield. LC-MS [M+H]+ 779.1927 m / z, observed 779. 1922 m / z.Scheme 3: Synthesis of GLPlR-SM-1286-p

[0182] Reagents and Conditions: (a) K2CO3, MeCN, 50°C, 34%. (b) 1 M 1,5,7-Triazabicyclo[4.4.0]-dec-5-ene(aq), MeCN followed by DIEA, COMU, azido-PEG4-amine.DCM, 60% over two steps.

[0183] Synthesis of 1161:

[0184] Compound 1161 was synthesized using General Procedure XI. The product was purified via flash chromatography (8% MeOH / DCM) to afford the product (34% yield) as a yellow solid. LC-MS(ES+): 609.1754 [M+H],

[0185] Synthesis of GLPlR-SM-1286-p:

[0186] GLP1R-SM-1286 was synthesized using General Procedure XII. The product was purified via flash chromatography (18% MeOH / DCM) to afford the product (60% yield over two steps) as a light yellow solid. MS(ES+): 839.52 [M+H],Scheme 4: Synthesis of GLPlR-SM-1295-p

[0187] Reagents and Conditions: (a) DIEA, MeCN, 87°C, 37%. (c) 1 M 1,5,7- Triazabicyclo[4.4.0]-dec-5-ene(aq), MeCN followed by DIEA. COMU, azido-PEG4-amine, DCM, 40% over two steps.

[0188] Synthesis of 1162:

[0189] To a stirred solution of methyl l-(2-(S-methylsulfonimidoyl)ethyl)-2- (trichloromethyl)-lH-benzo[d]imidazole-6-carboxylate (compound 1147, 0.100 g, 0.251 mmol) and 3-fluoro-4-(((5-(l,2,3,6-tetrahydropyridin-4-yl)thiazol-2- yl)oxy)methyl)benzonitrile (compound 1125, 0.237 g, 0.752 mmol) in MeCN (11 mL) was added N,N-diisopropylethylamine (0.44 mL, 2.51 mmol). The solution was heated to 87°C and stirred for 16 hours. An additional 5 equivalents of N,N-diisopropylethylamine were added and the reaction continued for an additional 24 hours. Volatiles were removed under reduced pressure and the crude material purified via flash chromatography (4% MeOH / DCM) to obtain the product as a light brown solid (0.057 g, 37% yield). LC-MS(ES+):623.15 [M+H],

[0190] Synthesis of GLPlR-SM-1295-p:

[0191] To a solution of methyl 2-(4-(2-((4-cyano-2-fluorobenzyl)oxy)thiazol-5-yl)-l,2,3,6- tetrahydropyridine-l-carbonyl)-l-(2-(S-methylsulfonimidoyl)ethyl)-lH-benzo[d]imidazole-6- carboxylate (compound 1162, 0.090 g, 0.145 mmol) in MeCN (1.45 mL) was added a 0.97 M aqueous solution of l,5,7-Triazabicyclo[4.4.0]-dec-5-ene (0.867 mL, 0.867 mmol). Thesolution stirred for 3 hours. The acidity' of the solution was adjusted to pH = 4 using 1 M HC1, then extracted three times with ethyl acetate. The organic layers were combined and dried over sodium sulfate. The volatiles were removed, then the crude material was dissolved in methylene chloride (1.45 mL). COMU (0.105 g, 0.246 mmol) and DIEA (0.13 mL, 0.723 mmol) were added, and the reaction stirred for 15 minutes, followed by addition of azido- PEG4-amine (0.057 g, 0.217 mmol). The reaction was stirred for 2 hours, then the volatiles were removed under reduced pressure. The crude material was purified via flash chromatography on silica gel (14% MeOEI / DCM) to afford GLP1R-SM-1295 (0.0597 g, 40% yield over two steps).Scheme 5. Synthesis of GLP-lR-SM21-p

[0192] Reagents and Conditions: (a) COMU, DIPEA. DCM, rt, 2 hr, 48%

[0193] Synthesis of GLP-lR-SM21-p:

[0194] GLP-1R-SM21 was synthesized following General Procedure 1. The product was purified using DCM: MeOH (96:4) to give an oil in 48% yield. LC-MS [M+H]+ 800.3895 m / z, observed 800.3893 m / z.Example 4. Synthesis of LipidsScheme 6. Synthesis of LP-161-p

[0195] Reagents and Conditions: (a) Mel, TEA, THF, rt, 2 h, 97%. (b) K2CO3, ACN, 85°C, 12 h, 73%. (c) TFA: DCM (3: 1). rt, 1 h, 94%. (d) TEA, DCM, rt, 12 h, 57%. (e) mCPBA, DCM, rt, 12 h, 76%.

[0196] Synthesis of 19:

[0197] To a solution of 4-(5-mercapto-l,3,4-oxadiazol-2-yl)phenol (1 eq) in anhydrous THF (0.2 M) was added TEA (1.2 eq) and Mel (1.1 eq) at 0°C under N2 gas. The mixture was allowed to warm to room temperature and stirred for 2 hours. After, the reaction was diluted with water (50 mL) and extracted thrice with ethyl acetate (3 x 50 mL). The combined organics were dried over Na2SC>4 and concentrated under reduced pressure. Product was obtained as an off-white solid by trituration with Et20 in 97% yield. LC-MS [M+H]+ 209.0385 m / z, observed 209.0389.

[0198] Synthesis of 110:

[0199] 110 as synthesized from 19 and N-Boc-PEG3-bromide following General Procedure II. The product was purified using hexanes: ethyl acetate (50:50) to give product as an oil in 73% yield. LC-MS [M+H]+ 484.2117 m / z, observed 484.2122.

[0200] Synthesis of Ill:

[0201] Il 1 was synthesized from 110 following General Procedure V to give product as an oil in 94% yield. LC-MS [M+H]+ 384.1593 m / z, observed 384.1585.

[0202] Synthesis of 112:

[0203] 112 was synthesized from II 1 & Palmitoyl chloride following General Procedure III.The product was purified using hexanes: ethyl acetate (30:70) to give product as an oil in 57% yield. LC-MS [M+H]+ 622.3890 m / z, observed 622.3884.

[0204] Synthesis of LP-161-p:LP-161 was synthesized from 112 following General Procedure IV. The product was synthesized using DCM: MeOH (96:4) to give an off-white solid in 76% yield. LC-MS [M+H]+ 654.3788 m / z, observed 654.3785.Scheme 7. Synthesis of LP-194-p

[0205] Reagents and Conditions: (a) COMU, DIPEA, DCM, rt, 2 h, 44%. (b) 25% piperidine in DMF. Rt. 1 h, 90%. (c) COMU, DIPEA, DCM, rt, 2 h, 54%. (d) TFA: DCM (3: 1), rt, 1 h, 96%.

[0206] Synthesis of E4:

[0207] E4 was synthesized following General Procedure I. The compound was purified using hexanes: ethyl acetate (80:20) to give product as an oil in 44% yield. LC-MS [M+H]+ 576.2710 m / z, observed 576.2709 m / z.

[0208] Synthesis of E5:

[0209] E5 was synthesized from E4 following General Procedure VII. The compound was purified using DCM: MeOH (96:4) to give product as an oil in 90% yield. LC-MS [M+H]+ 354.2029 m / z, observed 354.2030 m / z.

[0210] Synthesis of E6:

[0211] E6 was synthesized from E5 and 18-(tert-butoxy)-18-oxooctadecanoic acid following General Procedure I. The compound was purified using DCM: MeOH (96:4) to give product as an oil in 54% yield. LC-MS [M+H]+ 706.5006 m / z, observed 706.5008 m / z.

[0212] Synthesis of LP194-p:LP 194-p was synthesized from E6 following General Procedure V to give product as an off- white solid in 96% yield. LC-MS [M+H]+ 594.3754 m / z. observed 594.3753 m / z.

[0213] Reagents and Conditions: (a) K2CO3, ACN, 85°C, 12 h, 73%. (b) TFA: DCM (3: 1), rt, 1 h, 94%. (c) TEA, DCM. rt, 12 h, 57%. (d) mCPBA, DCM. rt, 12 h, 76%.

[0214] Synthesis of 113:

[0215] 113 was synthesized from 19 and tert-butyl (2-bromoethyl)carbamate followingGeneral Procedure II. The product was purified using hexanes: ethyl acetate (70:30) to give product as an oil in 73%. LC-MS [M+H]+ 352.1331 m / z, observed 352.1328.

[0216] Synthesis of 114:

[0217] 114 was synthesized from 113 following General Procedure V to give product as an oil in 94% yield. LC-MS [M+H]+ 252.0806 m / z, observed 252.0808.

[0218] Synthesis of 115:

[0219] 115 was synthesized from 114 & Cholesteryl chloride following General Procedure III. The product was purified using DCM: MeOH (98:2) to give product as an off-white solid in 57% yield. LC-MS [M+H]+ 664.4148 m / z, observed 664.4147.

[0220] Synthesis of LP-204-p:

[0221] LP-204 was synthesized from 115 following General Procedure IV. Product was purified using DCM: MeOH (96:4) to give an off-white solid in 76% yield. LC-MS [M+H]+ 696.4046 m / z, observed 696.4045.Scheme 9. Synthesis of LP-209-p

[0222] Reagents and Conditions: (a) TEA, DCM. rt, 12 h, 56% (b) mCPBA, DCM, rt, 12 h, 69%.

[0223] Synthesis of 116:

[0224] 116 was synthesized from 114 & Palmitoyl chloride following General Procedure III. The product was purified using hexanes: ethyl acetate (80:20) to give an off-white solid in 56% yield. LC-MS [M+H]+ 490.3103 m / z, observed 490.3100.

[0225] Synthesis of LP-209-p:

[0226] LP-209-p was synthesized from 116 following General Procedure IV. The product was purified using DCM: MeOH (98:2) to give an off-white solid in 69% yield. LC-MS [M+H]+ 522.3002 m / z, observed 522.3001.Scheme 10. Synthesis of LP-355-p

[0227] Reagents and Conditions: (a) COMU, DIPEA, DCM, rt, 2 h, 48%. (b) TFA: DCM (3: 1), rt, 1 h, 91%. (c) 25% piperidine in DMF. Rt. 1 h (d) TEA, THF, rt, 2 h, 57%.

[0228] Synthesis of E7 :

[0229] E7 was synthesized following General Procedure I. The product was purified using DCM: MeOH (96:4) to give an oil in 48% yield. LC-MS [M+H]+ 735.4948 m / z, observed 735.4949.

[0230] Synthesis of E8:

[0231] E8 was synthesized from E7 following General Procedure V to give product as an oil in 91% yield. LC-MS [M+H]+ 623.3696 m / z. observed 623.3698.

[0232] Synthesis of 117:

[0233] 117 was synthesized from E8 following General Procedure VII. The product was moved forward to the next step without any purification. LC-MS [M+H]+ 401.3015 m / z, observed 401.3017.

[0234] Synthesis of LP355-p:

[0235] LP355-p was synthesized from 117 & Sulfone TFP Ester following General Procedure III. The product was obtained using DCM: MeOH (96:4) to give an off-white solid in 57% yield. LC-MS [M+H]+ 651.3064 m / z. observed 651.3063.Scheme 11. Synthesis of LP-362-p

[0236] Reagents and Conditions: (a) TEA, DCM, rt, 3 h, 56% (b) TFA: DCM (3: 1), rt, 1 h, 93%. (c) COMU, DIPEA, DCM, rt, 2 h. 62% (d) TFA: DCM (3: 1). rt, 1 h, 96%. (e) TEA, THF, rt, 2 h, 49%.

[0237] Synthesis of E9:

[0238] E9 was synthesized from tert-butyl 3-aminobicyclo[l.l.l]pentane-l-carboxylate and palmitoyl chloride following General Procedure III. Product was obtained using hexanes: ethyl acetate (70:30) to give an oil in 56% yield. LC-MS [M+H]+ 422.3634 m / z. observed 422.3635.

[0239] Synthesis of 118:

[0240] 118 was synthesized from E9 following General Procedure V to give product as a white solid in 93% yield. LC-MS [M+H]+ 366.3008 m / z. observed 366.3010.

[0241] Synthesis of 119:

[0242] 119 was synthesized from 118 and N-Boc-Amino-PEG2- Amine following General Procedure I. Product was obtained using DCM: MeOH (98:2) to give an oil in 62% yield. LC-MS [M+H]+ 596.4639 m / z, observed 596.4638.

[0243] Synthesis of 120:

[0244] 120 was synthesized from 119 following General Procedure V to give product as an oil in 96% yield. LC-MS [M+H]+ 496.4114 m / z, observed 496.4113.

[0245] Synthesis of LP-362-p:LP-362 was synthesized from 120 following General Procedure III. Product was obtained using DCM: MeOH (96:4) to give an off-white solid in 49% yield. LC-MS [M+H]+ 746.4163 m / z, observed 746.4161.

[0246] To a 40 mL vial with stir bar and under N2 was added A-(2-aminospiro[3.3]hept-6- yl)carbamic acid tert-butyl ester (350 mg, 1.55 mmol), DCM (10 mL), and triethylamine (0.65 mL, 4.65 mmol). The reaction was stirred and cooled in an ice bath for 10 min and then palmitoyl chloride (0.51 mL, 1.7 mmol) was added dropwise over 30 seconds. A white precipitate immediately formed, and the reaction was stirred on ice for 10 minutes. The ice bath was removed and the reaction was stirred overnight, warming to room temperature. TLC (ninhydrin) confirmed reaction completion. The reaction was dry loaded with silica onto a 24 g silica gel column and purified with flash chromatography (EtOAc / hexanes, 0-70% over 35 min, ELS detection). After this duration, the column was flushed with 20% MeOH / DCM to expedite elution of product. Fractions were pooled and concentrated under reduced pressure to give 1 (602 mg) as a white solid in 84% yield. LC / MS (ESI+) calculated m / z 464.40 (M), found 465.64 (M + H+).

[0247] To a 40 mL vial with stir bar was added intermediate 1 (165 mg, 0.355 mmol) and 4 M HC1 in dioxanes (30 mL, 128.8 mmol). The vial was tightly capped and stirred for 2 h. The reaction immediately turned orange and then a white precipitate formed within 10 minutes. Reaction completion was confirmed by LCMS. The reaction was concentrated under reduced pressure, azeotroped with PhMe (5 mL), and dried overnight to give 2 (142 mg) as a white solid HC1 salt in quantitative yield. Intermediate 2 (142 mg. 0.354 mmol) was suspended in DCM (10 mL) and triethylamine (0. 15 mL, 1.06) and stir bar were added. NHS-PEG2- NHBoc (145 mg) was added last and the reaction was stirred for 2 h under N2. Reaction completion was confirmed by LCMS. The reaction was diluted with DCM and washed with saturated NaHCCh (x 2), brine, dried over MgSCh, filtered, and concentrated under reduced pressure to give crude material that was dissolved in DCM (8 mL) and dry loaded onto a 12 g silica column and purified with flash chromatography (MeOH / DCM 0-5%, ELS detection) to give intermediate 3 (172 mg) as a white solid in 78% yield. LC / MS (ESI+) calculated m / z 623.49 (M), found 624.78 (M + H+).

[0248] Intermediate 3 (170 mg, 0.272 mmol) was stirred in 4 M HC1 in dioxane (7 mL, 27.2 mmol) in a sealed RB flask for 2 h. LCMS confirmed reaction completion. The reaction was concentrated under reduced pressure, azeotroped with PhMe (5 mL), and dried overnight to give 4 (154 mg) as a white solid HC1 salt in quantitative yield. LC / MS (ESI+) calculated m / z 523.43 (M), found 524.60 (M + H+).To an oven dried 40 mL with stir bar and under N2 was added intermediate 4 (75 mg, 0. 135 mmol), sieve dried THF (3 mL), and triethylamine (0.12 mL, 0.815 mmol). 2, 3, 4,5,6- Pentafluorophenyl 4-[5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl]benzoate Sulfone (71 mg, 0.0163 mmol) was added and the reaction was stirred for 2 h. The reaction slowly became very heterogeneous. A small aliquot was taken, diluted with MeCN, and analyzed with LCMS to confirm reaction completion. The reaction was dry loaded with celite onto a 12 g silica gel column and purified with flash chromatography (MeOH / DCM 0-8% over 45 min, ELS detection) to give LP-361-p (52 mg) as a white solid in 52% yield. LC / MS (ESI+) calculated m / z 773.44 (M), found 775. 11 (M + H+).Scheme 13. Synthesis of LP-371-p

[0249] Compound 1 (palmitic acid, 2.50 g) was dissolved in 60 rnL DMF. Then TBTU (3.44 g) and DIPEA (6.9 mL) were added. The reaction was stirred for 10 minutes then compound 2 (2.66 g in DMF) was added. The reaction was complete after 1 hour. The mixture was diluted with 300 mL EtOAc and washed with 3% citric acid (3x60 mL), H2O (2x60 mL), and NaCl (1x60 mL), then dried over Na2SO4. The product was filtered and concentrated on rotary evaporator and high vacuum. The product was purified using column chromatography (loaded in DCM (15 mL) with a drop of MeOH onto a 80G RediSep Gold Rf column, mobile phase MeOH / DCM, 0-5% over 30 minutes. Yield 4.094 g. LC-MS: calculated [M+H] 486.74, found 488. 1 1.1

[0250] Compound 1 (4.094 g) was dissolved in 4M HCI in dioxane (28 mL) at 0°C for 10 minutes. The reaction was allowed to warm to room temperature then stirred for 2 hours. The product was concentrated on rotary evaporator and high vacuum. Yield 3.485 g. LC-MS: calculated [M+H] 386.57, found 388.02.Compound 1 (2.3 g) was dissolved in 80 mL THF. Then TEA (4.975 mL) and compound 2 (3.10 g) were added. The reaction was stirred for 1 hour. The reaction was dry loaded with Celite 545, the mixture was concentrated in a 28°C water bath and placed on high vacuum to fully dry. The product was purified with flash chromatography (MeOH / DCM, 0-6% over 40 min.) Yield 2.905 g. LC-MS: calculated [M+H] 636.85, found 637.95.Scheme 14. Synthesis of LP-389-p

[0251] Reagents and Conditions: (a) COMU, DIPEA. DCM. rt, 2 h, 47% (b) TFA: DCM (3: 1), rt, 1 h, 93%.

[0252] Synthesis of E10:

[0253] E10 was synthesized from E5 and 1 l-(4-(tert-butoxycarbonyl)phenoxy)undecanoic acid following General Procedure I. Product was obtained using DCM: MeOH (98:2) to give an oil in 47% yield. LC-MS [M+H]+ 714.4330 m / z, observed 714.4329.

[0254] Synthesis of LP389-p:

[0255] LP389-p was synthesized from E10 following General Procedure V to give product as an off-white solid in 93% yield. LC-MS [M+H]+ 602.3077 m / z, observed 602.3079.Scheme 15. Synthesis of LP-400-p

[0256] Reagents and Conditions: (a) TEA, THF, rt, 3 h, 63%

[0257] Synthesis of LP-400-p:

[0258] LP-400 was synthesized from Sulfone TFP Ester and hexadecan- 1 -amine following General Procedure III. The product was obtained using DCM: MeOH (98:2) to give an off- white solid in 63% yield. LC-MS [M+H]+ 492.2896 m / z, observed 492.2897.Scheme 16. Synthesis of LP-415-p

[0259] Reagents and Conditions: (a) TEA, THF, rt, 12 h, 54%. (b) TFA: DCM (3: 1), rt, 1 h, 95%. (c) TEA, THF, rt, 3 h, 41%.

[0260] Synthesis of 123:

[0261] 123 was synthesized from Sulfone TFP Ester and N-Boc-Amino-PEG3 -Amine following General Procedure III. The product was isolated using DCM: MeOH (96:4) to give an oil in 54% yield. LC-MS [M+H]+ 543.2125 m / z. observed 543.2124.

[0262] Synthesis of 124:

[0263] 124 was synthesized from 123 following General Procedure V to give product as an oil in 95% yield. LC-MS [M+H]+ 443.1600 m / z, observed 443.1603.

[0264] Synthesis of LP-415:

[0265] LP-415 was synthesized from 125 & Cholesteryl chloride following GeneralProcedure III. The product was isolated using DCM: MeOH (96:4) to give an off-white solid in 41% yield. LC-MS [M+H]+ 855.4942 m / z, observed 855.4941.Scheme 17. Synthesis of LP-416-p

[0266] Reagents and Conditions: (a) TEA, THF, rt, 3 h, 53%.

[0267] Synthesis ofLP-416-p:

[0268] LP416-p was synthesized from 124 & palmitoyl chloride following General ProcedureIII. The product was isolated using DCM: MeOH (96:4) to give an off-white solid in 53% yield. LC-MS [M+H]+ 695.4054 m / z, observed 695.4052.Scheme 18. Synthesis of LP-476-p

[0269] Reagents and Conditions: (a) TEA, THF, rt, 3 h, 84%

[0270] Synthesis of LP-476-p:

[0271] LP-473 was synthesized from Amino-PEG4-Azide and palmitoyl chloride following General Procedure III. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 84% yield. LC-MS [M+H]+ 501.4016 m / z, observed 501.4015.Scheme 19. Synthesis of LP-477-p

[0272] Reagents and Conditions: (a) NaN?. DMF, 60°C, 12 h, 62%

[0273] Synthesis of LP-477-p:

[0274] To a round bottom flask containing 1 -bromohexadecane (1 eq) in DMF (0.2 M) was added sodium azide (1.22 eq). The reaction was stirred at 60°C for 12 hours. After, the reaction was allowed to cool to room temperature and poured into cold water (20 mL). This solution was extracted thrice with ethyl acetate (3 x 30 mL). The combined organics were washed once with water (10 mL), once with brine (10 mL), dried over NazSC . and concentrated under reduced pressure to give product as an oil in 84% yield. LC-MS [M+H]+ 268.2753 m / z, observed 268.2758.Scheme 20. Synthesis of LP-481-p

[0275] Reagents and Conditions: (a) COMU, DIPEA, DCM, rt, 2 h, 61% (b) TFA: DCM (3: 1), rt, 1 h, 95%. (c) TEA, DCM, rt, 12 h, 39%. (d) mCPBA, DCM, rt, 12 h, 81%.

[0276] Synthesis of 123:

[0277] 123 was synthesized from II 1 and 6-((tert-butoxycarbonyl)amino)spiro[3.3]heptane-2- carboxylic acid following General Procedure I. Product was isolated using hexanes: ethyl acetate (70:30) to give an oil in 61% yield. LC-MS [M+H]+ 621.2958 m / z, observed 621.2960.

[0278] Synthesis of 124:

[0279] 124 was synthesized from 123 following General Procedure V to give product as an oil in 95% yield. LC-MS [M+H]+ 521.2434 m / z, observed 521.2433.

[0280] Synthesis of 125:

[0281] 125 was synthesized from 124 and palmitoyl chloride following General Procedure III. The product was isolated using DCM: MeOH (96:4) to give an oil in 39% yield. LC-MS [M+H]+ 759.4730 m / z, observed 759.4732.Synthesis of LP-481-p:

[0282] LP-481 was synthesized from 125 following General Procedure IV. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 81% yield. LC-MS [M+H]+ 791.4629 m / z, observed 791.4628.Scheme 21. Synthesis of LP-482-pReagents and Conditions: (a) COMU, DIPEA, DCM. rt, 2 h, 58% (b) TFA: DCM (3: 1), rt, 1 h, 95%. (c) TEA, DCM, rt, 12 h, 44%. (d) mCPBA, DCM, rt, 12 h, 76%.

[0283] Synthesis of 126:

[0284] 126 was synthesized from Il l and 3-((tert- butoxycarbonyl)amino)bicyclo[l. l. l]pentane-l-carboxylic acid following General Procedure I. Product was isolated using hexanes: ethyl acetate (70:30) to give an oil in 58% yield. LC- MS [M+H]+ 593.2645 m / z, observed 593.2642.

[0285] Synthesis of 127:

[0286] 127 was synthesized from 126 following General Procedure V to give product as an oil in 95% yield. LC-MS [M+H]+ 493.2121 m / z. observed 493.2120.

[0287] Synthesis of 128:

[0288] 128 was synthesized from 127 and palmitoyl chloride following General Procedure III. The product was isolated using DCM: MeOH (96:4) to give an oil in 44% yield. LC-MS [M+H]+ 731.4417 m / z, observed 731.4419.

[0289] Synthesis of LP-482-p

[0290] LP-482 was synthesized from 128 following General Procedure IV. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 76% yield. LC-MS [M+H]+ 763.4316 m / z, observed 763.4315.Scheme 22. Synthesis of LP-484-p

[0291] Reagents and Conditions: (a) TEA, DCM, rt, 12 h, 41%. (b) mCPBA, DCM, rt, 12 h, 71%.

[0292] Synthesis of 129:

[0293] 129 was synthesized from II 1 and docosanoyl chloride following General Procedure III. Product was isolated using DCM: MeOH (96:4) to give an oil in 41% yield. LC-MS [M+H]+ 706.4829 m / z, observed 706.4828.

[0294] Synthesis of LP-484:

[0295] LP-484 was synthesized from 129 following General Procedure IV. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 71% yield. LC-MS [M+H]+ 738.4727 m / z, observed 738.4728.Scheme 23. Synthesis of LP-485-p

[0296] Reagents and Conditions: (a) COMU, DIPEA, DCM, rt, 2 h, 51% (b) TFA: DCM (3: 1). rt, 1 h, 95%. (c) TEA, DCM, rt, 12 h, 40%. (d) mCPBA, DCM, rt, 12 h, 69%.

[0297] Synthesis of 130:

[0298] 130 was synthesized from Il l and 4-((tert- butoxycarbonyl)amino)bicyclo[2.2.2]octane-l-carboxylic acid following General Procedure I. Product was isolated using hexanes: ethyl acetate (70:30) to give an oil in 51% yield. LC- MS [M+H]+ 635.3115 m / z, observed 635.3114.

[0299] Synthesis of 131:

[0300] 131 was synthesized from 130 following General Procedure V to give product as an oil in 95% yield. LC-MS [M+H]+ 535.2590 m / z, observed 535.2589.

[0301] Synthesis of 132:

[0302] 132 was synthesized from 131 and palmitoyl chloride following General Procedure III. The product was isolated using DCM: MeOH (96:4) to give an oil in 40% yield. LC-MS [M+H]+ 773.4887 m / z, observed 773.4886.

[0303] Synthesis of LP-485-p:

[0304] LP-485 was synthesized from 132 following General Procedure IV. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 69% yield. LC-MS [M+H]+ 805.4785 m / z, observed 805.4788.Scheme 24. Synthesis of LP-516-p

[0305] Reagents and Conditions: (a) H2, Pd / c, EtOAc, rt, 12 h, 97% (b) TEA, DCM, rt, 12 h, 30%. (c) LiOH, MeOH: H2O (2: 1), rt, 3 h, 94%. (d) COMU, DIPEA, DCM, rt, 12 h, 53%.

[0306] Synthesis of 133:

[0307] Methyl 1.4-dibenzyl-l,4-diazepane-6-carboxylate was dissolved in EtOAc (0.2 M) and purged for 15 min with nitrogen gas. Then, a catalytic amount of Pd / c was added. The reaction was purged twice with a hydrogen balloon for 15 min each and then allowed to run overnight at room temperature under hydrogen gas. After, the reaction mixture was fdtered over celite to obtain product as an oil in 97% yield. LC-MS [M+H]+ 159.1134 m / z, observed 159.1138.

[0308] Synthesis of 134:

[0309] 134 was synthesized from 133 and palmitoyl chloride following General Procedure III. The product was isolated using hexanes: ethyl acetate (60:40) to give an oil in 30% yield. LC-MS [M+H]+ 635.5727 m / z, observed 635.5721.

[0310] Synthesis of 135:

[0311] 135 was synthesized from 134 following General Procedure VI to give product as an off-white solid in 94% yield. LC-MS [M+H]+ 621.5570 m / z, observed 621.5575.

[0312] Synthesis of LP-516-p:

[0313] LP-516 was synthesized from 135 and 2-(2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4- oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)ethoxy)ethan-l -amine following General Procedure I. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 53% yield. LC- MS [M+H]+ 1018.6878, observed 1018.6877.Scheme 25. Synthesis of LP-517-p

[0314] Reagents and Conditions: (a) TEA, DCM, rt, 12 h, 28%. (b) TFA: DCM (3: 1), rt, 1 h, 95%. (c) COMU, DIPEA, DCM, rt, 12 h, 48%.

[0315] Synthesis of 136:

[0316] 136 was synthesized from N-(t- butyl ester-PEG3)-N-bis(PEG3-amine) and palmitoyl chloride following General Procedure III. Product was isolated using DCM: MeOH (96:4) as an oil in 28% yield. LC-MS [M+H]+ 1132.8927 m / z, observed 1132.8925.

[0317] Synthesis of 137:

[0318] 137 was synthesized from 136 following General Procedure V to give product as an oil in 95% yield. LC-MS [M+H]+ 1076.8301 m / z, observed 1076.8300.

[0319] Synthesis of LP-517:

[0320] LP-517 was synthesized from 137 and 2-(2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4- oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)ethoxy)ethan-l -amine following General Procedure I. Product was isolated using DCM: MeOH (96:4) to give an off-white solid in 48% yield. LC- MS [M+H]+ 1473.9608 m / z, observed 1473.9610.Scheme 26. Synthesis of LP-518-pLP518-p

[0321] Reagents and Conditions: (a) TEA, DCM, rt, 12 h, 25%. (b) 25% piperidine in DMF, rt, 1 h, 90%. (c) K2CO3, ACN. 85°C, 12 h. 64%. (d) mCPBA. DCM, rt. 12 h. 60%. (e) TFA: DCM (3: 1), rt, 1 h, 96%. (f) COMU, DIPEA, DCM, rt, 12 h, 53%.

[0322] Synthesis of 138:

[0323] 138 was synthesized from (9H-fluoren-9-yl)methyl bis(14-amino-3,6,9,12- tetraoxatetradecyl)carbamate and palmitoyl chloride following General Procedure III. Product was isolated using DCM: MeOH (96:4) to give an oil in 25% yield. LC-MS [M+H]+ 1154.8559 m / z, observed 1154.8564.

[0324] Synthesis of 139:

[0325] 139 was synthesized from 138 following General Procedure VII. Product was isolated using DCM: MeOH (95:5) to give an oil in 90% yield. LC-MS [M+H]+ 932.7878 m / z. observed 932.7871.

[0326] Synthesis of 140:

[0327] 140 was synthesized from 19 following General Procedure II. Product was isolated using hexanes: ethyl acetate (50:50) to give an oil in 64% yield. LC-MS [M+H]+ 455. 1852 m / z, observed 455.1858.

[0328] Synthesis of 141:

[0329] 141 was synthesized from 140 following General Procedure IV. Product was isolated using hexanes: ethyl acetate (30:70) to give an off-white solid in 60% yield. LC-MS [M+H]+ 487. 1750 m / z, observed 487. 1755.

[0330] Synthesis of 142:

[0331] 142 was synthesized from 141 following General Procedure V to give product as an off-white solid in 96% yield. LC-MS [M+H]+ 431.1124 m / z, observed 431.1129.

[0332] Synthesis of LP-518-p:

[0333] LP-518 was synthesized from 142 and 139 following General Procedure I. Product was isolated using DCM: MeOH (96:4) to give an oil in 53% yield. LC-MS [M+H]+ 1344.8819 m / z, observed 1344.8811.Example 5. Conjugation of Linking Groups and Targeting Ligands to RNAi agents

[0334] A. Conjugation of Activated Ester Linking Groups

[0335] The following procedure was used to conjugate linking groups having the structure of L4 as shown in Table 1 above to an RNAi agent with an amine-functionalized sense strand, such as C6-NH2, NH2-C6, or (NH2-C6)s, as shown in Table 1, above. An annealed RNAi Agent driedby lyophilization was dissolved in DMSO and 10% water (v / v%) at 25 mg / mL. Then 50-100 equivalents of TEA and 3 equivalents of activated ester linker were added to the solution. The solution was allowed to react for 1-2 hours, while monitored by RP-HPLC-MS (mobile phase A 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile on an Waters™ XBridge Cl 8 column, Waters Corp.)

[0336] The product was then precipitated by adding 12 mL acetonitrile and 0.4 mL PBS and centrifuging the solid to a pellet. The pellet was then re-dissolved in 0.4 mL of 1XPBS and 12 mL of acetonitrile. The resulting pellet was dried on high vacuum for one hour.

[0337] B. Conjugation of Targeting Ligands to Propargyl Linkers

[0338] Either prior to or after annealing, the 5' or 3' tri dentate alkyne functionalized sense strand is conjugated to the GLP1R Ligands. The following example describes the conjugation of GLP1R ligands to the annealed duplex: Stock solutions of 0.5M Tris(3- hydroxypropyltriazolylmethyljamine (THPTA), 0.5M of Cu(II) sulfate pentahydrate (Cu(II)SO4 5 H2O) and 2M solution of sodium ascorbate were prepared in deionized water. A 75 mg / mL solution in DMSO of GLP1R ligand was made. In a 1.5 mL centrifuge tube containing tri-alkyne functionalized duplex (3mg, 75pL, 40mg / mL in deionized water, approximately 15,000 g / mol), 25 pL of IM Hepes pH 8.5 buffer is added. After vortexing, 35 pL of DMSO was added and the solution is vortexed. GLP1R ligand was added to the reaction (6 eq / duplex, 2 eq / alkyne, approximately 15pL) and the solution is vortexed. Using pH paper, pH was checked and confirmed to be pH approximately 8. In a separate 1.5 mL centrifuge tube, 50 pL of 0.5M THPTA was mixed with l OuL of 0.5M Cu(II)SO4 5 H2O, vortexed, and incubated at room temp for 5 min. After 5 min, THPTA / Cu solution (7.2 pL, 6 eq 5: 1 THPTA:Cu) was added to the reaction vial, and vortexed. Immediately afterwards, 2M ascorbate (5 pL, 50 eq per duplex. 16.7 per alkyne) was added to the reaction vial and vortexed. Once the reaction was complete (typically complete in 0.5-1 h), the reaction mixture was immediately purified by non-denaturing anion exchange chromatography.Example 6. Conjugation of lipid PK / PD modulator precursors

[0339] Either prior to or after annealing and prior to or after conjugation of one or more targeting ligands, one or more lipid PK / PD modulator precursors can be linked to the RNAi agents disclosed herein. The following describes the general conjugation process used to link lipid PK / PD modulator precursors to the constructs set forth in the Examples depicted herein.

[0340] A. Conjugation of a maleimide-containing lipid PK / PD modulator precursor

[0341] The following describes the general process used to link a maleimide-containing lipid PK / PD modulator precursor to the (C6-SS-C6) or (6-SS-6) functionalized sense strand of an RNAi agent by undertaking a dithiothreitol reduction of disulfide followed by a thiol-Michael Addition of the respective maleimide-containing lipid PK / PD modulator precursor: In a vial, functionalized sense strand was dissolved at 50mg / mL in sterilized water. Then 20 equivalents of each of 0.1 M Hepes pH 8.5 buffer and dithiothreitol were added. The mixture was allowed to react for one hour, then the conjugate was precipitated in acetonitrile and PBS, and the solids were centrifuged into a pellet.

[0342] The pellet was brought up in a 70 / 30 mixture of DMSO / water at a solids concentration of 30 mg / mL. Then, the maleimide-containing lipid PK / PD modulator precursor was added at 1.5 equivalents. The mixture was allowed to react for 30 minutes. The product was purified on an AEX-HPLC (mobile phase A: 25 mM TRIS pH=7.2, 1 mM EDTA, 50% acetonitrile; mobile phase B: 25 mM TRIS pH=7.2, 1 mM EDTA, 500 mM NaBr, 50% acetonitrile; solid phase TSKgel-30; 1.5 cmxlO cm.) The solvent was removed by rotary evaporator, and desalted with a 3K spin column using 2x10 mL exchanges with sterilized water. The solid product was dried using lyophilization and stored for later use.

[0343] B. Conjugation of a sulfone-containing lipid PK / PD modulator precursor

[0344] In a vial, functionalized sense strand was dissolved at 50mg / mL in sterilized water. Then 20 equivalents of each of 0.1M Hepes pH 8.5 buffer and dithiothreitol are added. The mixture was allowed to react for one hour, then the conjugate was precipitated in acetonitrile and PBS, and the solids were centrifuged into a pellet.

[0345] The pellet was brought up in a 70 / 30 mixture of DMSO / water at a solids concentration of 30 mg / mL. Then, the sulfone-containing lipid PK / PD modulator precursor was added at 1.5 equivalents. The vial was purged with N2, and heated to 40°C while stirring. The mixture was allowed to react for one hour. The product was purified on an AEX-HPLC (mobile phase A: 25 mM TRIS pH=7.2, 1 mM EDTA, 50% acetonitrile; mobile phase B: 25 mM TRIS pH=7.2, 1 mM EDTA, 500 mM NaBr, 50% acetonitrile; solid phase TSKgel-30;1.5 cmxlO cm.) The solvent was removed by rotary evaporator, and desalted with a 3K spin column using 2x10 mL exchanges with sterilized water. The solid product was dried using lyophilization and stored for later use.

[0346] C. Conjugation of an azide-containing lipid PK / PD modulator precursor

[0347] One molar equivalent of TG-TBTA resin loaded with Cu(I) was weighed into a glass vial. The vial was purged with N2 for 15 minutes. Then, functionalized sense strand was dissolved in a separate vial in sterilized water at a concentration of 100 mg / mL. Then twoequivalents of the azide-containing lipid PK / PD modulator precursor (50 mg / mL in DMF) is added to the vial. Then TEA, DMF and water are added until the final reaction conditions are 33 mM TEA, 60% DMF, and 20 mg / mL of the conjugated product. The solution was then transferred to the vial with resin via a syringe. The N2 purge was removed and the vial was sealed and moved to a stir plate at 40°C. The mixture was allowed to react for 16 hours. The resin was filtered off using a 0.45 pm filter.

[0348] The product was purified using AEX purification (mobile phase A: 25 mM TRIS pH=7.2, ImM EDTA, 50% acetonitrile; mobile phase B: 25mM TRIS pH=7.2, ImM EDTA, 500mM NaBr, 50% acetonitrile solid phase TSKgel-30; 1.5 cmxlO cm.) The acetonitrile was removed using a rotary evaporator, and desalted with a 3K spin column using 2x10 mL exchanges with sterilized water. The solid product was dried using lyophilization and stored for later use.

[0349] D. Conjugation of an alkyne-containing lipid PK / PD modulator precursor

[0350] The following describes the general process used to link an activated alkyne- containing lipid PK / PD modulator precursor to the (C6-SS-C6) or (6-SS-6) functionalized sense strand of an RNAi agent by undertaking a dithiothreitol reduction of disulfide followed by addition to an alkyne-containing PK / PD modulator precursor: In a vial, 10 mg of siRNA comprising the (C6-SS-C6) or (6-SS-6) functionalized sense strand was dissolved at 50 mg / mL in sterilized water. Then 20 equivalents of each of 0.1M Hepes pH 8.5 buffer and dithiothreitol (IM in sterilized water) were added. The mixture was allowed to react for one hour, then purified on Waters™ XBridge BEH C4 Column using a mobile phase A of lOOmM HFIP, 14 mM, and TEA, and a mobile phase B of Acetonitrile using the following formula, wherein %B indicates the amount of mobile phase B while the remainder is mobile phase A.

[0351] The product was precipitated once by adding 12 mL of acetonitrile and 0.4mL 1XPBS, and the resulting solid was centrifuged into a pellet. The pellet was re-dissolved in 0.4 mL 1XPBS and 12 mL of acetonitrile. The pellet was dried on high vacuum for one hour.

[0352] The pellet was brought up in a vial a 70 / 30 mixture of DMSO / water at a solids concentration of 30 mg / niL. Then, the alkyne-containing lipid PK / PD modulator precursor was added at 2 equivalents relative to siRNA. Then 10 equivalents of TEA was added. The vial was purged using N2, and the reaction mixture was heated to 40°C while stirring. The mixture was allowed to react for one hour. The product was purified using anion-exchange HPLC using a TSKgel-30 (available from Tosoh Biosciences) packed column, 1.5cm x 10 cm. using a mobile phase A of 25mM TRIS pH=7.2, ImM EDTA, 50% Acetonitrile, and a mobile phase B of 25mM TRIS pH=7.2, ImM EDTA, 500mM NaBr, 50% Acetonitrile using the following formula, wherein %B indicates the amount of mobile phase B while the remainder is mobile phase A.

[0353] The fractions containing the product were collected, and acetonitrile was removed using a rotary evaporator. The product was desalted with a 3K spin column, using 2 x 10 mL exchanges with sterilized water. The product was then dried using lyophilization and stored for later use.

[0354] Table 2: RNAi Agents used in the following examples:a = 2'-O-methyladenosine-3'-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3 '-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3 '-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate is = 2'-O-methylinosine-3'-phosphorothioate t = 2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us = 2'-O-methyluridine-3'-phosphorothioateAf = 2'-fluoroadenosine-3'-phosphateAfs = 2'-fluoroadenosine-3'-phosporothioateCf = 2'-fluorocytidine-3'-phosphateCfs = 2'-fluorocytidme-3'-phosphorothioateGf = 2'-fluoroguanosine-3'-phosphateGfs = 2'-fluoroguanosine-3'-phosphorothioateUf = 2'-fluorouridine-3 '-phosphateUfs = 2'-fluorouridine-3'-phosphorothioate a _2N = see Table 1 a_2Ns = see Table 1(invAb) = inverted abasic deoxy ribonucleotide-5'- phosphate, see Table 1 (invAb)s = inverted abasic deoxy ribonucleotide-5 '- phosphorothioate, see Table 1 s = phosphorothioate linkage ss = phosphorodithioate linkageOther structures, see Table 1Example 7. In Vivo Knockdown of SOD 1 in Rats

[0355] On study day 1, male Sprague Dawley rats were dosed subcutaneously with either saline solution or test article comprising LP161 and SM1295 conjugated to an siRNA targeting rat SOD1 formulated in saline, according to the dosing schedule below:

[0356] Table 3: Dosing regimen for the rats of Example 7.

[0357] On study day 2, three rats from group 4 were sacrificed. On study days 15, 29, 43, 57, 85 and 113, three rats from each group were sacrificed and samples of heart tissue were taken from each animal. Samples were analyzed by qPCR for SOD1 mRNA knockdown, using rat ARL1 (rARLl) as a control gene. Treatment groups were normalized to group 1 (saline control). Average knockdown of SOD1 for each group are shown in Table 4 below:

[0358] Table 4: Relative expression of SOD1 mRNA in heart tissue analyzed by qPCR for each of the dosing groups of Example 7.

[0359] As can be seen in Table 4, a dose-response was observed for animals injected with AC008676 through day 113. Furthermore, animals dosed with AC008676 continued to show deep knockdown well after the animals were dosed, even at the 1 mpk dose level.Example 8. In Vivo Knockdown of SOI) 1 in Cynomolgus Monkeys

[0360] On Study day 1, cynomolgus monkeys were subcutaneously injected with either saline or test article of a conjugate using lipids described herein conjugated to an siRNA targeting cyno SOD1 formulated in saline, according to Table 5 below:

[0361] Table 5: Dosing regimen for the monkeys of Example 8.

[0362] On study day 36, animals were sacrificed and tissue was collected from heart apex, left ventricle, right ventricle, left atrium and right atrium. Samples were analyzed by qPCR for SOD1 mRNA knockdown, using cynomolgus ARL1 (cARLl) as a control gene.Treatment groups were normalized to group 1 (saline control). Average knockdown of SOD1 for each group in the collected tissues are shown in Table 6 below:

[0363] Table 6: Relative expression of SOD1 mRNA in heart tissues analyzed by qPCR for each of the dosing groups of Example 8.

[0364] As can be seen in Table 6, RNAi agents comprising GLP1R ligands SM-4, SM-1286, and SM-1295 and LP-161 are delivered to heart tissue in cynomolgus monkeys. Each of the tissues analyzed showed deep knockdown of SOD1 at the 36-day time point.Example 9. In vivo Knockdown of Myh6 in mice

[0365] On study day 1, female Balb / c mice were dosed intravenously with either saline solution or test article comprising LP161 and SM4 or LP161 and SM19 conjugated to an siRNA targeting the mouse gene Myh6, formulated in saline, according to the dosing schedule below:

[0366] Table 7: Dosing regimen for the mice of Example 9.

[0367] On study days 8, 15, 29, 43, and 57, four mice from each group (except for day 57: 5 mice from groups 1 and 2 and three mice from group 3) were sacrificed and samples of heart tissue were taken from each animal. Samples were analyzed by qPCR for Myh6 mRNA knockdown, using mouse ARL1 (mARLl) as a control gene. Treatment groups were normalized to group 1 (saline control). Average knockdown of Myh6 for each group are shown in Table 8 below:Table 8: Relative expression of Myh6 mRNA in heart tissue analyzed by qPCR for each of the dosing groups of Example 9.

[0368] As can be seen in Table 8 both AC005927 (comprising GLP1R-SM-4) and AC006685 (comprising GLP1R-SM-19) showed deep and durable knockdown of a gene expressed in heart tissue through day 57.Example 10. In vivo Knockdown of SOD 1 in Mice

[0369] On study day 1, female Balb / c mice were dosed subcutaneously with either saline solution or test article, according to the dosing schedule below:

[0370] Table 9: Dosing regimen for the mice of Example 10.

[0371] On study day 8, mice were sacrificed and samples of heart tissue were taken from each animal. Samples were analyzed by qPCR for SOD1 mRNA knockdown, using mouse ARL1 (mARLl) as a control gene. Treatment groups were normalized to group 1 (saline control). Average knockdown of SOD1 for each group are shown in Table 10 below:

[0372] Table 10: Relative expression of SOD1 mRNA in heart tissue analyzed by qPCR for each of the dosing groups of Example 10.

[0373] As can be seen in Table 10, select RNAi agents comprising a lipid achieve deep and durable knockdow n of a gene expressed in heart tissue. Group 8, dosed with AC002645 (comprising LP-396 and LP-161) demonstrated the greatest knockdown of SOD1 at 0.5 mpk.Example 11. In vivo Knockdown ofSODl in Mice

[0374] On study day 1, female Balb / c mice were dosed intravenously (IV) with either saline solution or test article comprising a GLP1R targeting ligand, according to the dosing schedule below:

[0375] Table 11: Dosing regimen for the mice of Example 11.

[0376] On study day 8, mice were sacrificed and samples of heart tissue were taken from each animal. Samples were analyzed by qPCR for SOD1 mRNA knockdown, using mouse ARL1 (mARLl) as a control gene. Treatment groups were normalized to group 1 (saline control). Average knockdown of SOD1 for each group are shown in Table 12 below:

[0377] Table 12: Relative expression of SODl mRNA in heart tissue analyzed by qPCR for each of the dosing groups of Example 11.

[0378] As can be seen in Table 12 RNAi agents comprising a GLP1R targeting ligand and a lipid achieve deep and durable knockdown of a gene expressed in heart tissue. Group 2, dosed with AC002700 (comprising GLP1R-SM-4) demonstrated the greatest knockdown of SOD1 at 0.5 mpk.Example 12. In vivo Knockdown of SOD I in Mice

[0379] On study day 1, female Balb / c mice were dosed intravenously (IV) with either saline solution or test article comprising a GLP1R targeting ligand, according to the dosing schedule below:

[0380] Table 13: Dosing regimen for the mice of Example 12.

[0381] On study day 8, mice were sacrificed and samples of heart tissue were taken from each animal. Samples were analyzed by qPCR for SOD1 mRNA knockdown, using mouse ARL1 (mARLl) as a control gene. Treatment groups were normalized to group 1 (saline control). Average knockdown of SOD1 for each group are shown in Table 14 below :

[0382] Table 14: Relative expression of SOD1 mRNA in heart tissue analyzed by qPCR for each of the dosing groups of Example 12.

[0383] As can be seen in Table 14, RNAi agents comprising a GLP1R targeting ligand and a lipid achieve deep and durable knockdown of a gene expressed in heart tissue. Group 6, dosed with AC003390 (comprising GLP1R-SM-4) demonstrated the greatest knockdown of SOD1.Example 13. In vivo Knockdown ofSODl in Mice

[0384] On study day 1, female Balb / c mice were dosed intravenously (IV) with either saline solution or test article comprising a GLP1R targeting ligand, according to the dosing schedule below:

[0385] Table 15: Dosing regimen for the mice of Example 13.

[0386] On study day 8, mice were sacrificed and samples of heart tissue were taken from each animal. Samples were analyzed by qPCR for SOD1 mRNA knockdown, using mouse ARL1 (mARLl) as a control gene. Treatment groups were normalized to group 1 (saline control). Average knockdown of SOD1 for each group are shown in Table 16 below:

[0387] Table 16: Relative expression of SOD1 mRNA in heart tissue analyzed by qPCR for each of the dosing groups of Example 13.

[0388] As can be seen in Table 27, RNAi agents comprising a GLP1R targeting ligand and a lipid achieve deep and durable knockdown of a gene expressed in heart tissue. Group 5, dosed with AC003392 (comprising GLP1R-SM-21) demonstrated the greatest knockdown of SOD1 at 0.3 mpk.EQUIVALENTS AND SCOPE

[0389] In the claims articles such as “a,” "an.” and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherw ise relevant to a given product or process.

[0390] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g, in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0391] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict betw een any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particularembodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Claims

1. CL IMSWhat is claimed is:wherein,R comprises an oligonucleotide-based agent;Li is a linking moiety;L2 is selected from the group consisting of: optionally substituted alkylene, optionally substituted arylene, 2-20 polyethylene glycol (PEG) units optionally interrupted by C(O)NRa, wherein R3 is selected from Ci-Ce alkyl and H, and a bond;Z is selected from the group consisting of: CH (wherein p is 1). CH2 (wherein p is 0), N (wherein p is 1), optionally substituted arylene, C(O)NRs (wherein p is 0), heterocyclene, and NR3 (wherein p is 0), wherein R3 is selected from Ci-Ce alkyl and H; p is 0 or 1, as valency permits;Yi and Y2 are each independently selected from the group consisting of: 2-20 polyethylene glycol (PEG) units, optionally substituted alkyl, optionally substituted cycloalkyl, and a bond;Xi and X2 are each independently selected from the group consisting of: C(O), C(O)NR2 and a bond, wherein R2 is Ci-Ce alkyl or H; n and m are each independently an integer from 8 to 20;Wi and W2 are each independently selected from the group consisting of: H, COOH2. The compound of claim 1, wherein Li is selected from the group consisting of -S-,,The compound of claim 1 or claim 2, wherein Li is5. The compound of any one of claims 1-4. wherein L2 is selected from the group6. The compound of any one of claims 1-5, wherein Z is selected from the group consisting of:bond.

7. The compound of any one of claims 1-6, wherein Y i is selected from the group8. The compound of any one of claims 1-5. wherein Xi is C(O)NR2.

9. The compound of claim 6, wherein R2 is H.

10. The compound of any one of claims 1-7, wherein n is 13, 14, 15, or 1 .

11. The compound of any one of claims 1-8, wherein p is 0.

12. The compound of any one of claims 1-10, wherein p is 1.

13. The compound of claim 12, wherein Z is N.

14. The compound of any one of claims 1-13, wherein Wi is H.

15. The compound of any one of claims 1-14, wherein R is an RNAi agent.

16. A compound, or a pharmaceutically acceptable salt thereof, selected from the group17. A compound, or a pharmaceutically acceptable salt thereof, of the formula:

19. A compound selected from the group consisting of:

20. A method of delivering an RNAi agent to a cardiomyocyte, comprising administering to a subject the compound of any one of claims 1-17.

21. A method of inhibiting the expression of a gene expressed in a cardiomyocyte, comprising administering to a subject the compound of any one of claims 1-17.

22. The compound of any one of claims 1-17, for use in inhibiting expression of a gene expressed in a cardiomyocyte.

23. A method of making a compound of Formula I:wherein,R comprises an oligonucleotide-based agent;Li is a linking moiety;L2 is selected from the group consisting of: optionally substituted alkylene, optionally substituted arylene, 2-20 polyethylene glycol (PEG) units optionally interrupted by C(O)NRa, wherein R3 is selected from Ci-Ce alkyl and H, and a bond;Z is selected from the group consisting of: CH (wherein p is 1), CH2 (wherein p is 0), N (wherein p is 1), optionally substituted arylene, C(O)NRs (wherein p is 0), heterocyclene, and NR3 (wherein p is 0), wherein R3 is selected from Ci-Ce alkyl and H; p is 0 or 1, as valency permits;Yi and Y2 are each independently selected from the group consisting of: 2-20 polyethylene glycol (PEG) units, optionally substituted alkyl, optionally substituted cycloalkyl, and a bond;Xi and X2 are each independently selected from the group consisting of: C(O), C(O)NR2 and a bond, wherein R2 is Ci-Ce alkyl or H; n and m are each independently an integer from 8 to 20;Wi and W2 are each independently selected from the group consisting of: H, COOH andcomprising reacting a compound of claim 19 with an RNAi agent comprising a reactive group to form the compound of Formula I.

24. The method of claim 23, wherein the reactive group is selected from the group consisting of: disulfide, amine, and alkyne.

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