Novel RNA therapeutics and uses thereof

Abstract

The present invention relates to novel therapeutic compounds, known as RNAi agents, that decrease expression of the ANGPTL8 receptor (expressed by the ANGPTL8 gene), thereby decreasing expression of mRNA and protein expression. Such RNAi agents are useful in the treatment of diseases involving the regulation of ANGPTL8 expression and function, such as dyslipidemia, a cardiovascular disorder, or a cardiometabolic disorder.

Description

NOVEL RNA THERAPEUTICS AND USES THEREOFSEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “31120_WO” created 30-May-2025 and is 3.59 megabytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention is directed to nucleic acids, including RNA molecules such as therapeutic RNA molecules, such as RNAi agents targeting ANGPTL8 mRNA, comprising a novel internucleotide linkage, termed a “2-propyl” linkage. Also disclosed herein are nucleotides and intermediates for making the same. These “2-propyl” nucleotides are termed “PrON” herein. The present invention is also directed to novel nucleosides comprising a PrON moiety. The present invention is also directed to oligonucleotides, such as RNAi agents, comprising at least one PrON.BACKGROUND

[0003] The present disclosure relates to novel RNAi agents designed to decrease the expression of ANGPTL8 in the liver, where the RNAi agents comprise delivery moieties conjugated to oligonucleotides optionally via a linker. The RNAi agents are useful in the treatment of diseases involving the regulation of ANGPTL8 expression.

[0004] Angiopoietin-like protein 8 (ANGPTL8) is mainly expressed in liver and adipose tissue and it plays an important role in triglyceride metabolism. ANGPTL8, together with ANGPTL3 or ANGPTL4, is thought to regulate triglyceride levels by inhibiting the enzymatic activity of lipoprotein lipase (LPL), which, when active, hydrolyzes triglycerides 10 and decreases circulating plasma triglycerides. Increased levels of ANGPTL8 arc observed or associated with cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), aberrant renal function, hypertension, nonalcoholic fatty liver disease such as nonalcoholic steatohepatitis (NASH), and obesity.

[0005] ANGPTL8 siRNAs and ASOs have been described, such as those disclosed in W02020 / 104649 A2, but none have progressed for treatment in patients. Using the ANGPTL8 RNAi agents herein to decrease expression of ANGPTL8 can be employed, e.g., to treat cardiometabolic and related disorders such as dyslipidemia, in patients in need thereof.

[0006] As RNA is relatively unstable, much effort has been expended in balancing the stability of potential therapeutic RNA molecules against sequence-specific knockdown capability, with any cellular or organ toxicity. Organisms including humans harbor numerous threats to RNA durability, particular RNase H family ribonucleases, which degrade both natively-produced and exogenous RNA molecules. Low durability of RNA can result in low exposure of the therapeutic oligonucleotide to its intended target.

[0007] Stability-enhancing modifications available in the art include phosphorothioate (PS) internucleotide linkages and extended nucleic acid (exNA) as described in International Patent Publication No. WO2021 / 195533. As the field advances, however, more options for extending the half life of therapeutic oligonucleotides, such as RNAi agents is needed. In particular, modifications which reduce or eliminate degradation artifacts are desired.SUMMARY

[0008] In one aspect, provided herein are RNAi agents for reducing ANGPTL8 gene expression, wherein the RNAi agent comprises at least one nucleotide including a modified intcmuclcotidc linkage. In one aspect, the present disclosure provides an oligonucleotide of Formula I:wherein:R1is selected from the group consisting of H, OR2, NH2, SR2, F, and Cl, wherein R2is selected from the group consisting of H and C1-C20 alkyl; andB is a nucleobase.

[0009] In one embodiment, the present disclosure provides a double- stranded RNA molecule which includes a first oligonucleotide as described above, and a second oligonucleotide of 15 to 30 nucleotides in total length, wherein the first oligonucleotide and the second oligonucleotide form a duplex region. In one aspect, the first oligonucleotide is an antisense strand of an RNAi agent, and the second oligonucleotide is a sense strand of an RNAi agent.

[0010] In some embodiments, the RNAi agent comprises a delivery moiety of Formula II conjugated to R, wherein R is a double stranded RNA (dsRNA) comprising an antisense strand and a sense strand:Formula II, wherein R is conjugated to connection point E of Formula II, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises a region of complementarity to an ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense and antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified intemucleotide linkages. In some embodiments, Formula II is conjugated to the sense strand, optionally via a linker. In some embodiments, Formula II is conjugated to the 3’ terminal nucleotide of the sense strand, optionally via a linker.

[0011] In an embodiment, provided herein are RNAi agents for reducing ANGPTL8 gene expression, wherein the RNAi agent comprises a delivery moiety of Formula II conjugated toR, wherein R is a double stranded RNA (dsRNA) comprising an antisense strand and a sense strand:Formula II, wherein R is conjugated to connection point E of Formula II, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a sequence having 90% sequence identity thereto, or an antisense strand sequence as set forth in Tables 3A, 3B, and 4, or a sequence having 90% sequence identity thereto, and wherein the sense and antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified internucleotide linkages. In some embodiments, Formula II is conjugated to the sense strand, optionally via a linker. In some embodiments, Formula II is conjugated to the 3’ terminal nucleotide of the sense strand, optionally via a linker.

[0012] In some embodiments, the antisense strand is 15 to 50 nucleotides in length. In some embodiments, the sense strand is 15 to 50 nucleotides in length. In some embodiments, the antisense strand is between 18 and 23 nucleotides in length. In some embodiments, the sense strand is between 18 and 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

[0013] In some embodiments, the sense strand or the antisense strand comprises a sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5 disclosed herein. In some embodiments, the sense strand and the antisense strand comprise a sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5 disclosed herein.

[0014] In some embodiments, R is conjugated to Formula II via a linker. In further embodiments, the linker comprises a linker of Formula III having connection points A and B or the linker comprises Formula IV having connection points X and Y, and wherein:Formula IV; a. Formula II, at connection point E, conjugated to Formula III at connection point A and Formula III is conjugated to a phosphate group at connection point B, and the phosphate group is further conjugated to R; or b. Formula II, at connection point E, conjugated to Formula IV at connection point X and Formula IV is conjugated to a phosphate group at connection point Y, and the phosphate group is further conjugated to R.

[0015] In embodiments, the ANGPTL8 RNAi agent includes at least one 2-propyl nucleotide (PrON) as described herein.

[0016] In another aspect, provided herein are pharmaceutical composition comprising the ANGPTL8 RNAi agent described herein and one or more pharmaceutically acceptable excipients.

[0017] In another aspect, provided herein are methods of treating cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), aberrant renal function, hypertension, nonalcoholic fatty liver disease such as nonalcoholic steatohepatitis (NASH),or obesity in a patient in need thereof, comprising administering to the patient a ANGPTL8 RNAi agent or pharmaceutical composition thereof described herein.

[0018] In another aspect, provided herein are ANGPTL8 RNAi agent for use in a therapy.Also provided herein are uses of ANGPTL8 RNAi agent in the manufacture of a medicament for the treatment of cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), aberrant renal function, hypertension, nonalcoholic fatty liver disease such as nonalcoholic steatohepatitis (NASH), or obesity.DETAILED DESCRIPTION

[0019] Such siRNAs may exhibit one or more of, e.g., as compared to other liver targeted siRNAs such as ANGPTL8 siRNAs comprising a different delivery ligand, a different sequence, a differently modified sequence, or as compared to treatment with a vehicle control: improved knockdown in the liver; improved tissue exposure, improved exposure in liver hepatocytes; an improved durable response; an improved pharmacokinetic profile; fewer off target effects; and / or an improved toxicity profile. Other embodiments of the ANGPTL8 RNAi agents herein may include one or more of fewer side effects as compared to statins or other standard of care; an improved toxicity profile; an improved safety profile; improved tolerability or compliance; and / or improved liver function tests. Still other siRNAs herein may have other benefits, e.g., in combination with any of the preceding or as a standalone benefit, including improved and / or simplified synthesis, synthetic processes with fewer degradation products; or any combination thereof.

[0020] The RNAi agents herein comprise a sense strand and an antisense strand, wherein each is an oligonucleotide. In some embodiments, the RNAi agent described herein also comprises a delivery moiety. As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase such as, for example, adenine, cytosine, guanine, thymine, or uracil; and a pentose sugar such as, for example, ribose or 2'-deoxyribose) and a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0021] As used herein, “oligonucleotide” means a short nucleic acid compound (e.g., less than about 100 nucleotides in length). An oligonucleotide may be single- stranded (ss) or double stranded (ds). An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (DsiRNA), or antisense oligonucleotide (ASO).

[0022] As used herein, “ribonucleotide” means a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0023] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified intemucleotide linkage can be a non- naturally occurring linkage.

[0024] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxy ribonucleotide. A modified nucleotide can be a non-naturally occurring nucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[0025] The term “percentage sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment forpurposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, Clustal W2.0 or Clustal X2.0 or Megalign (DNASTAR) software. In one embodiment herein, sequence identity is calculated use Clustal W2.0 or Clustal X2.0. In another embodiment, sequence identity is calculated using Clustal W2.0. In another embodiment, sequence identity is calculated using Clustal X2.0. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase 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. The output is the percent identity of the subject sequence with respect to the query sequence. In some embodiments, percent sequence identity is the percent of nucleotide residues that are identical between two strands using the PID3 calculation, which is the number of identical nucleotide residues divided by the total number of nucleotides of the shortest of the two sequences, multiplied by 100. See, e.g., Raghava, G., Barton, G.J. Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006).

[0026] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5' terminal nucleotide of an oligonucleotide in place of a 5'-phosphate. A 5' phosphate analog can include a phosphatase-resistant linkage. Examplesof phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'- VP). An oligonucleotide can have a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”) at a 5'-terminal nucleotide. An example of a 4'-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'- carbon) or analog thereof. See, e g., Inti. Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., Inti. Patent Application No. WO 2011 / 133871; US Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0027] As used herein, “region of complementarity” means a nucleotide sequence of a nucleic acid (e.g., a double stranded oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, an oligonucleotide herein includes a targeting sequence having a region of complementary to a mRNA target sequence.

[0028] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, such as a sense strand or an antisense strand means a structure formed through hydrogen bonds of complementary base pairing of two antiparallcl sequences of nucleotides under suitable conditions to promote such a structure. A duplex may form despite not having full complementarity between the two strands, or when an abasic nucleotide is present. A Duplex No:, as shown herein, e.g., in Table 2A, Table 2B, Table 3A Table 3B, or Table 4 corresponds to a specific sense and antisense strand that comprise a given RNAi agent.

[0029] RNA interference is a specialized cellular process that utilizes RISC for degrading RNA in a sequence dependent manner. As used herein, “RNAi agent” comprises either (a) a double stranded oligonucleotide having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a single stranded oligonucleotide having a single antisense strand, where that antisense strand (or part of thatantisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA. In some embodiments, RNAi agent comprises a delivery moiety.

[0030] As used herein, the term “control agent” refers to an RNAi agent which does not appreciably decrease levels of ANGPTL8 transcripts or expression.

[0031] As used herein, a bond illustrated asindicates a connection point as described therein. For example, if a generic variable, e.g., X, is stated to be attached at the connection point E as shown below, this is intended to show X is bonded to the atom of the connection point (see the scheme below).X is bonded at connection point E O

[0032] As used herein, “treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, and need not indicate a total elimination of all disorder or disease symptoms. Treatment includes administration of an RNAi agent or pharmaceutical composition thereof for treatment of a disease or condition in a mammal including a human.

[0033] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a RNAi agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the RNAi agent to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the RNAi agent are outweighed by the therapeutically beneficial effects.

[0034] Provided herein are RNAi agents for reducing ANGPTL8 gene expression, wherein the RNAi agent comprises a delivery moiety of Formula II conjugated to R, wherein R is a double stranded RNA (dsRNA) comprising an antisense strand and a sense strand:Formula II, wherein R is conjugated to connection point E of Formula 11, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises a region of complementarity to a ANGTPL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense and antisense strand each optionally comprise one or more modified nucleotides and one or more modified intemucleotide linkages.

[0035] Also provided here are RNAi agents for reducing ANGPTL8 gene expression, wherein the RNAi agent comprises a delivery moiety of Formula Ila conjugated to R, wherein R comprises an antisense strand and a sense strand:Formula Ila, wherein R is optionally conjugated to Formula Ila via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises a region of complementarity to a ANGPTL8 mRNA target sequence of SEQ IDNO: 511, and wherein the sense and antisense strand each optionally comprise one or more modified nucleotides and one or more modified intemucleotide linkages.

[0036] Disclosed herein are RNAi agents for reducing ANGPTL8 gene expression, wherein the RNAi agents comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises at least 15 nucleotides as set forth in an antisense strand sequence disclosed herein, and wherein the sense strand and / or the antisense strand each optionally comprise one or more modified nucleotides and / or modified internucleotide linkages. In further embodiments, the antisense strand comprises at least 15 nucleotides of an antisense strand sequence in Table 2A, Table 2B, Table 3 A, Table 3B, Table 4, or Table 5. In further embodiments, the RNAi agent reduces ANGPTL8 gene expression by about 50% or greater in a cell expressing ANGPTL8, as compared to a control. In further embodiments, the RNAi agent reduces ANGPTL8 gene expression by reducing the level of ANGPTL8 mRNA transcript, the level of ANGPTL8 protein, or both.

[0037] In further embodiments, the antisense strand is 15 to 25 nucleotides in length, and / or the sense strand is 15 to 25 nucleotides in length. In further embodiments, the antisense strand is between 18 and 23 nucleotides in length. In further embodiments, the sense strand is between 18 and 21 nucleotides in length.

[0038] In further embodiments, the RNAi agent comprises at least 18 contiguous nucleotides of an antisense strand sequence set forth in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5.

[0039] In further embodiments, the antisense strand of the RNAi agent is 23 nucleotides in length. In still further embodiments, the sense strand is 21 nucleotides in length. In another embodiment, the sense and antisense strand comprise a sequence selected from the sequences set forth in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5.

[0040] The sense strand and the antisense strand of the RNAi agents disclosed herein do not require full complementarity. Accordingly, in the RNAi agents disclosed herein, the duplex region between the sense strand and the antisense strand comprises 0, 1, 2, or 3 mismatches between the sense strand and the antisense strand. In further embodiments, the duplexregion between the sense strand and the antisense strand consists of 0, 1, 2, or 3 mismatches between the sense strand and the antisense strand.

[0041] In one embodiment, the antisense strand of an ANGPTL8 RNAi agent includes at least one PrON.

[0042] In one embodiment, the sense strand of an ANGPTL8 RNAi agent includes at least one PrON.

[0043] In further embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides, such as 2’ fluoro modified nucleotides or 2’-O- methyl modified nucleotides. In one aspect, the oligonucleotide may include at least one modification selected from the group consisting of 2’-O-methoxy, 2’-O-methyl, 2’-fluoro, phosphorothioate, 2’-deoxy, abasic moiety, inverted abasic moiety, glycol nucleic acid (GNA), extended nucleic acid (exNA), R-PrON (that is PrON in which the branched methyl of the internucleotide linkage is in the R configuration), and S-PrON (that is, PrON in which the branched methyl of the internucleotide linkage is in the S configuration); that is, R-PrON includes the following structure:and S-PrON includes the following structure:

[0044] In some embodiments, an extended nucleic acid (or exNA) moiety can be included in at least one of the sense strand and the antisense strand. The exNA-containing nucleic acid can be prepared as described in WO2021 / 195533. The exNA has the following formula:As used in experiments herein, the nucleobase B of exNA is uracil.

[0045] In still further embodiments of the RNAi agents disclosed herein, each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide.

[0046] In further embodiments, each nucleotide is a 2’ fluoro modified nucleotide or a 2’-O- methyl modified nucleotide.

[0047] In one aspect, the oligonucleotide as provided herein includes PrONs in nonconsecutive positions. In an example of an oligonucleotide having two PrONs in such a fashion is provided as Formula X:X.In this formula, Xe is one or more nucleotides.

[0048] The skilled artisan will be capable of envisioning formulae for oligonucleotides including three, four, or five PrONs, in view of Formula X above.

[0049] In further embodiments, the antisense strand has a sequence as set forth in an antisense strand sequence in Table 2A, Table 2B, Table 4, or Table 5, or a sequence having at least 90% sequence identity thereto, or an antisense strand sequence in Table 3A or Table 3B, or a sequence having at least 90% sequence identity thereto. In other embodiments, theantisense strand sequence or the sense strand sequence in Table 2A or Table 2B or Table 3A or Table 3B or Table 4 or Table 5 is independently a sequence that is at least 80, 85, 90, or 95, percent identical thereto.

[0050] In further embodiments of the RNAi agents disclosed herein, the antisense strand is 23 nucleotides in length, each nucleotide of the antisense strand is a modified nucleotide, and the 2’ fluoro modified nucleotides may appeal- at different positions than is shown in the sequences in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5. In an embodiment, the 2’ Fluoro modified nucleotides are present at a. Positions 2, 3, 7, 14, and 16 from the 5’ end of the antisense strand; or b. Positions 2, 5, 7, 14, and 16 from the 5’ end of the antisense strand; or c. Positions 2, 3, 8, 14, and 16 from the 5’ end of the antisense strand; or d. Positions 2, 5, 8, 14, and 16 from the 5’ end of the antisense strand; or e. Positions 2, 6, 14, and 16 from the 5’ end of the antisense strand; or f. Positions 2, 14, and 16 from the 5’ end of the antisense strand.In further embodiments, the nucleotides that are not 2’ fluoro modified nucleotides are 2’ 0- methyl modified nucleotides.

[0051] In further embodiments of the RNAi agents disclosed herein, the sense strand and antisense strand each independently comprise one or more modified intcmuclcotidc linkages, and each modified internucleotide linkage is a phosphorothioate linkage. In further embodiments, the sense strand and antisense strand each independently comprise four phosphorothioate linkages. In still further embodiments, the two terminal nucleotides at each of the 5’ and 3’ ends of each of the sense and antisense strand are phosphorothioate linkages.

[0052] In other embodiments, the 5’ nucleotide of the antisense strand comprises a naturally occurring OH group, or is modified to contain a phosphate group or a phosphate analog. As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5' terminal nucleotide of an oligonucleotide in place of a 5'-phosphate. A 5' phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate(5'-MP) and 5'-(E)-vinylphosphonate (5'- VP). An oligonucleotide can have a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”) at a 5'- terminal nucleotide. An example of a 4'-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its d'carbon) or analog thereof. See, e g., Inti. Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., Inti. Patent Application No. WO 2011 / 133871; US Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0053] In further embodiments, the 5’ terminal nucleotide of the antisense strand may comprise a further modification, wherein the 5’ terminal nucleotide contains as a 5’ a vinyl phosphonate, a phosphate, or a hydroxyl group. In other embodiments, the phosphate group listed at the 5’ end of the recited SEQ ID NO: is removed and replaced with an OH. In other embodiments, the phosphate group listed at the 5’ end of the recited SEQ ID NO: is replaced with a 5’ vinylpho sphonate.

[0054] In further embodiments, 1, 2, or 3 mismatches are introduced into the sense strand sequence of Table 2A Table 2B, Table 3A, Table 3B, Table 4, or Table 5. In further embodiments, 1 , 2, or both terminal nucleotides of 5’ end of the antisense strand are changed.

[0055] In some embodiments of the RNAi agents herein, the antisense strand comprises a first nucleic acid sequence that has at least 90% sequence identity to an antisense sequence corresponding to a Duplex NO: in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5 and the sense strand comprises a second nucleic acid sequence that has at least 90% sequence identity to a sense sequence corresponding to the same Duplex No: in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5. For example, in one embodiment, the antisense strand comprises a first nucleic acid sequence that has at least 90% sequence identity to an antisense sequence corresponding to a Duplex NO: 1 in Table 2A, that is, a first nucleic acid sequence that has at least 90% sequence identity to SEQ ID NO:6, and the sense strand comprises a second nucleic acid sequence that has at least 90% sequence identity to a sense sequence corresponding to Duplex No: 1 in Table 2A, that is, SEQ ID NO: 1. Infurther embodiments, the 5’ phosphate of the antisense strand is further modified / replaced, and is a 5’ vinylpho sphonate or an OH group.

[0056] In further embodiments, the 5’ terminal nucleotide of the antisense strand is substituted such that the final sequence contains a vinylphosphonate, a phosphate group, or an OH group.

[0057] In other embodiments disclosed herein are RNAi agents having a delivery moiety of Formula II conjugated to R:Formula II, wherein R is a dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that have complementarity to ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense strand and the antisense strand form a region of complementarity of at least 15 nucleotides, and wherein the sense strand and antisense strand are each independently 18 to 23 nucleotides in length, and optionally wherein the sense strand and antisense strand each independently comprise one or more modified nucleotides, and optionally wherein the sense strand and the antisense strand each independently comprise one or more modified intemucleotide linkages, and wherein R is optionally conjugated to Formula II via a linker. In further embodiments, the sense or the antisense strand is selected from a Table 2A, Table 2B, Table 3A, Table 3B, Table 4, or Table 5 disclosed herein. In other embodiments, the antisense or antisense strand of the RNAi agent has an antisense strand sequence and / or a sense strand sequence of at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding sequence selected from a Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 herein.

[0058] In other embodiments, the RNAi agent disclosed herein comprises a linker. In other further embodiments R is conjugated to Formula II via a linker. In further embodiments, the linker comprises a linker of Formula 111 having connection points A and B or the linker comprises Formula IV having connection points X and Y, and wherein:Formula IV ; a. the RNAi agent comprises Formula II, at connection point E, conjugated to FormulaIII at connection point A and Formula III is conjugated to a phosphate group at connection point B, and the phosphate group is conjugated to R; or b. the RNAi agent comprises Formula II, at connection point E, conjugated to FormulaIV at connection point X and Formula IV is conjugated to a phosphate group at connection point Y, and the phosphate group is further conjugated to R.

[0059] In other embodiments wherein he RNAi agent comprises a linker, R is conjugated to Formula II via a linker, and the linker is a linker comprising Formula IV having connection points X and Y :Formula IV ; and wherein the RNAi agent comprises Formula I conjugated to Formula IV at connection point X and Formula IV is conjugated to a phosphate group at connection point Y, and the phosphate group is further conjugated to R.

[0060] The sense strand and antisense strand of RNAi agent can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), lohn Wiley & Sons, Inc., New York, NY, USA), H- phosphonatc, phosphortricstcr chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from Bio Automation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides.

[0061] In still other embodiments, the RNAi agent is capable of decreasing expression of the ANGPTL8 gene in a liver cell. In other embodiments, the RNAi agents disclosed herein are for use in therapy. In further embodiments, the use is for the treatment of dyslipidemia such as high plasma triglyceride levels. In other embodiments, the RNAi agents disclosed herein are for use in the treatment of cardiovascular disease. In other embodiments herein, the RNAi agents are for use in preventing a cardiovascular event. In further embodiments, the cardiovascular event is myocardial infarction. In other embodiments, the use is for decreasing hospitalizations related to cardiovascular disease or events. In other embodiments, the use is for treating non-alcoholic fatty liver disease (NAFLD). In further embodiments,wherein the NAFLD is non-alcoholic steatohepatitis (NASH). In other embodiments, the use is for decreasing inhibition of lipoprotein lipase (LPL). In further embodiments, the use is for increasing catabolism of triglyceride rich lipoproteins. In other embodiments, the RNAi agents are for use in treating a liver disease in a patient that would benefit from decreasing expression levels of ANGPTL8. In other embodiments, the use is for treatment of any of the preceding, after statin use failed to control one or more symptoms, e.g. failed to reduce one or more of elevated total-C, LDL-C, apo B, and / or failed to increase HDLC. In other embodiments, the use is for the treatment of any of the preceding, in patients that are statin intolerant; in further embodiments, the use is for lowering LDL-C in patients that are statin intolerant. In further embodiments, the use is for any of the preceding uses, after alteration of diet failed to control one or more symptoms. In other further embodiments, the use is for any of the preceding uses, as an adjunct therapy to diet.

[0062] The RNAi agents may be formulated into pharmaceutical compositions.Accordingly, disclosed herein are pharmaceutical compositions comprising the RNAi agent disclosed herein, and one or more pharmaceutically acceptable excipients. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).

[0063] In other embodiment are uses of the RNAi agents herein for the manufacture of a medicament for the treatment of dyslipidemia or any of the uses recited in the preceding paragraph.

[0064] In other embodiments are methods of treating dyslipidemia, in patients in need thereof, comprising administering an RNAi agent disclosed herein, or a pharmaceutical composition thereof, to the patient. In other embodiments are methods of treating dyslipidemia, or any of the uses recited above, in patients in need thereof, or a pharmaceutical composition thereof. In other embodiments are methods of treating a patient unable to achieve lipid levels following statin and / or diet therapy, comprising administering to the patient an RNAi agent disclosed herein, or a pharmaceutical composition thereof.

[0065] The RNAi agent can be administered to the patient intravenously or subcutaneously.

[0066] RNAi dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, severaldivided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0067] Dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0068] In other embodiments are methods of decreasing ANGPTL8 expression in a cell, comprising contacting the cell with an RNAi agent disclosed herein, and incubating the cell for a time sufficient for decreasing the level of ANGPTL8 mRNA by at least 50% as compared to an untreated or control treated cell.

[0069] Disclosed herein are oligonucleotides including at least one PrON, including singlestranded RNA molecules, double- stranded RNA molecules, and RNAi agents. The 2-propyl internucleotide linker represents a departure from natural internucleotide linkages and as such, may provide an RNA molecule in which it is included with lesser susceptibility to degradation by nucleases, and in turn, when used in the context of a therapeutic molecule, can possess higher durability than an oligonucleotide which does not include a PrON, and better exposure to target tissue. In some instances, the resistance to degradation conferred by PrON can reduce the number and type of metabolic byproducts (fragmented RNA molecules).

[0070] In certain embodiments, an oligonucleotide of the present disclosure has one PrON. In another embodiment, the oligonucleotide has two PrONs. In another embodiment, the oligonucleotide includes three PrONs, or four PrONs, or five PrONs. A PrON may be incorporated at the 5’ end of an oligonucleotide in one embodiment. In another embodiment, the PrON is incorporated or at the 3’ end of the oligonucleotide. In another embodiment, the PrON is incorporated at an internal portion of the oligonucleotide. A PrON may be included in a duplex region of a double-stranded RNA in one embodiment. In another embodiment, the PrON or PrONs may be present in an overhang portion of the dsRNA. In one embodiment, the PrON-containing oligonucleotide is, or is a portion of, an ANGPTL8 RNAi agent.

[0071] In an oligonucleotide with multiple PrONs, the PrONs can be located in consecutive positions, or may instead be spaced apart (that is, separated by at least one non-PrON nucleotide.)

[0072] The introduction of the 2-propyl internucleotide linker gives rise to a stereocenter. In some instances, the PrON introduced into an oligonucleotide has the R stereochemical configuration. In another embodiment, the PrON included in an oligonucleotide has the S stereochemical configuration. In an embodiment wherein the oligonucleotide has multiple PrONs, the stereochemical configuration of each PrON is selected independently. For example, in an oligonucleotide containing two PrONs, the configuration of the two PrONs in a 5’ to 3’ direction may be R / R, R / S, S / R, or S / S. In one instance, such an oligonucleotide has these two PrONs in consecutive positions, in some embodiments as a 3’ overhang in the antisense strand of a dsRNA duplex.

[0073] The PrON as envisaged herein can be used in an oligonucleotide with other modified intemucleotide linkers, including but not limited to phosphorothioate linkers. The PrON can be used with other modified nucleotides as disclosed herein.

[0074] In an embodiment, Xi is selected from hydroxyl, phosphate and vinyl phosphonate. In certain embodiments, Xi may be a 5’ end cap moiety. In some embodiments, Xi may simply indicate the 5’ end of the oligonucleotide; that is, it may be considered to be absent, or simply be a spatial designator for the end of the strand.

[0075] In an embodiment, X4 may be a 3’ end cap moiety. In an embodiment, X4 may be a linker or a targeting group. In such embodiments, Q may be PO2X5. In some embodiments, X4 may simply indicate the 3’ end of the oligonucleotide; that is, it may be considered to be absent, or simply be a spatial designator for the end of the strand.

[0076] Further provided herein is a pharmaceutical composition for use as a therapy, or for treatment of a disease or a condition, which includes an oligonucleotide as described herein. In one embodiment, the pharmaceutical composition may include a salt of the oligonucleotide. RNA is a polyanion owing to the negative charges present on the phosphate groups of the backbone, and these charges may be balanced by provision of positive charges, yielding a salt of the RNA. In one instance, the salt is a sodium salt. In another instance, thesalt is a potassium salt. In another embodiment, the salt may include multiple different cationic species.PREPARATIONS

[0077] The compounds of the present disclosure may be prepared by following the schemes and preparations detailed below. These schemes and preparations are not limiting in their scope.

[0078] Certain abbreviations are defined as follows: “AS” refers to antisense strand; “CT” refers to cycle threshold; “DCM” refers to dichloromethane; “DMF” refers to dimethylformamide; “DIEA” refers to N,N-diisopropylethylamine; “PE” refers to petroleum ether; “TFA” refers to trifluoroacetic acid; “DMT” refers to dimethoxytrityl; “DMTQ” refers to dimethoxytrityl chloride; “DMSO” refers to dimethyl sulfoxide; “dsRNA” refers to double stranded ribonucleic acid; “EtOH” refers to ethanol; “EtOAc” refers to ethyl acetate; “hiPSC” refers to human induced pluripotent stem cell; “MeCN” refers to acetonitrile; “MeOH” refers to methanol and methyl alcohol; “PBS” phosphate-buffered saline; “PCR” refers to polymerase chain reaction; “RT-PCR” refers to reverse transcription polymerase chain reaction; “siRNA” refers to small interfering RNA; “SS” refers to sense strand; “TEA” refers to tricthylaminc; “IBX” refers to 2-iodoxybcnzoic acid “9-BBN” refers to 9- borabicyclo [3.3.1] nonane; “2, 4, 6 TMP” refers to 2, 4, 6 trimethylpyridine; “TBAF” refers to tetra-butylammonium fluoride; “TBDMSC1” refers to tert-butyldimethylsilyl chloride; “9- BBN” refers to 9-borabicyclo[3.3.1]nonane; “CAP A” refers to capping agent; “CPG” refers to controlled pore glass; “CV” refers to column volume; “1,2-DCE” refers to 1,2- dichloroethane; “DIEA” refers to N,N-diisopropylethylamine; “DMAP” refers to 4- dimethylaminopyridine; “DMF” refers to N,N-dimethylformamide; “EDCI HQ” refers to 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; “ES / MS” refers to electrospray mass spectrometry; “Et2O” refers to diethyl ether; “GalNAc” refers to N- acetylgalactosamine; “HATU” refers to l-[bis(dimethylamino)methylene]-lH-l,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; “HBTU” refers to O-(benzotriazol-l- yl)-A,A,A',A'-tetramethyluronium hexafluorophosphate; “HOBt” refers to 1-hydroxybenzotriazole hydrate; “IBX” refers to 2-iodoxybenzoic acid; “LDHA” refers to lactate dehydrogenase-A; “MeCN” refers to acetonitrile; “MWCO” refers to molecular weight cut-off; “NHS” refers to N-hydroxysuccinimide; “NMR” refers to nuclear magnetic resonance; “OD” refers to optical density; “PBS” refers to phosphate-buffered saline; “PE” refers to petroleum ether; “TFA” refers to trifluoro acetic acid; “THF” refers to tetrahydrofuran; “TEC” refers to thin line chromatography; and “TMP” refers to 2, 2,6,6- tetramethylpiperidine; and “TBDMS” refers to tert-butyldimethylsilyl.

[0079] Scheme 1, step A depicts the cyclization of compound (1) using trimethylsilyl trifluoromethanesulfonate in a solvent such as 1,2-DCE to give compound (2).

[0080] Scheme 2, Step A depicts the condensation of compound (3) with compound (4) using a suitable base such as NaOH in a solvent such as DMSO to give compound (5). Step B shows the protection of compound (5) with benzyl chloroformate using a base such assodium carbonate in a solvent such as DCM to give compound (6). Step C shows the hydrolysis of compound (6) with formic acid to give triacid compound (7).Scheme 3

[0081] Scheme 3, Step A depicts the protection of compound (8) with benzyl bromide using a base such as potassium carbonate in a solvent such as acetone to give compound (9). Step B shows the O-alkylation of compounds (9) and (10) using tetrabutylammonium sulfate and sodium hydroxide to give compound (11). Step C shows the debenzylation of compound (11) using a suitable catalyst such as palladium hydroxide on carbon and hydrogen gas in a solvent such as MeOH to give compound (12).

[0082] Scheme 4, Step A depicts the amide coupling between compounds (7) and (12) using HBTU and a base such as DIEA in a solvent such as DMF to give compound (13). Step B shows the acidic deprotection of compound (13) with p-toluenesulfonic acid monohydrate in a solvent such as MeOH to give compound (14). Step C shows the addition of compound (14) to compound (2) using trimethylsilyl trifluoromethanesulfonate in a solvent such as 1,2- DCE to give compound (15). Step D shows the deprotection of compound (15) using a catalyst such as palladium on carbon and a gas such as hydrogen in a solvent such as MeOH to give compound (16). Step E shows the amide coupling between compounds (16) and (17) using HBTU and a base such as DIEA in a solvent such as DMF to give compound (18). Step F shows the hydrogenation of compound (18) using palladium on carbon in a solvent such as MeOH to give compound (19).Scheme 5

[0083] Scheme 5, step A depicts the protection of compound (20) using DMTC1 with a suitable base such as DIEA in a solvent such as DCM to give compound (21). Step B shows an amide coupling between compound (21) and piperidin-4-yl methanol using HBTU and HOBt with TMP in a solvent such as DCM to give compound (22). The deprotection of compound (22) with 20% piperidine in DMF to give compound (23) is shown in step C.Scheme 6

[0084] Scheme 6, step A depicts an amide coupling between compounds (19) and (23) using HBTU and HOBt with an appropriate base such as DIEA in a solvent such as DMF to give compound (24). Step B shows the formation of compound (25) by adding succinic anhydride to compound (24) in an appropriate solvent such as DCM with a base system of TEA and DMAP. Step C depicts the loading of compound (25) onto resin with 2-(lH-benzotriazol-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate and a base such as DIEA in a solvent system such as MeCN and DCM to give compound (26).Scheme 728

[0085] Scheme 7, Step A depicts a coupling reaction between compounds (19) and (27) using a reagent such as EDCI HC1 in a solvent such as DCM to give compound (28).

[0086] Scheme 8, step A depicts the synthesis of compound (30) by DMT protection of compound (29), the conditions of which will be known by one skilled in the art. Step B shows the conversion of compound (30) to compound (31) by protection of the 3’ alcohol using TBDMSC1 in an appropriate solvent such as DMF. For step C, the DMT group was removed from compound (31) to give compound (32), the conditions of which will be known by one skilled in the ait. Step D depicts the oxidation of compound (32), using IBX in an appropriate solvent such as EtOAc, to provide compound (33). Conversion of compound (33) to compound (34) via a Wittig reaction, in step E, was accomplished using methyltriphenylphosphonium bromide in an appropriate solvent such as THF.Hydroboration of compound (34) in step F, using 9-BBN in an appropriate solvent such as THF, gave compound (35) which was then oxidized in step G, using IBX and an appropriatesolvent such as MeCN, to provide compound (36). A Grignard reaction, depicted in step H, was used to convert compound (36) to compound (37) using methylmagnesium bromide in an appropriate solvent such as THF. DMT protection of compound (37), depicted in Step I, provided compound (38) using DMTC1, 2, 4, 6-TMP, and AgNCh in an appropriate solvent such as DCM. The TBDMS group was removed from compound (38) to provide compound (39), the conditions of which will be known by one skilled in the art.Scheme 9

[0087] Scheme 9, step A depicts the SFC purification of compound (39) to give compounds (40) and (41) as diastereomers. In step B, the 3’ alcohol of compound (40) was phosphitylated with 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile in an appropriate solvent such as DCM to provide compound (42). Step C, using the same conditions as step B, provided compound (43) from compound (41).Scheme 10

[0088] Scheme 10, steps A-C depict the formation of compound (47) through several steps beginning with compounds (19) and (44). The steps are essentially analogous to those found in Scheme 6, steps A-C.Scheme 11

[0089] Scheme 11, step A depicts the synthesis of compound (2) by DMT protection of compound (1), the conditions of which will be known by one skilled in the ait. Step B shows the conversion of compound (2) to compound (3) by protection of the 3’ alcohol using TBDMSC1 in an appropriate solvent such as DMF. For step C, the DMT group was removed from compound (3) to give compound (4), the conditions of which will be known by one skilled in the ait. Step D depicts the oxidation of compound (4), using IBX in an appropriate solvent such as EtOAc, to provide compound (5). Conversion of compound (5) to compound (6) via a Wittig reaction, in step E, was accomplished using methyltriphenylphosphonium bromide in an appropriate solvent such as THF. Hydroboration of compound (6) in step F, using 9-BBN in an appropriate solvent such as THF, gave compound (7) which was then oxidized in step G, using IBX and an appropriate solvent such as ACN, to provide compound(8). A Grignard reaction, depicted in step H, was used to convert compound (8) to compound (9) using methylmagnesium bromide in an appropriate solvent such as THF. DMT protection of compound (9), depicted in Step I, provided compound (10) using DMTC1, 2, 4, 6-TMP, and AgNCh in an appropriate solvent such as DCM. The TBDMS group was removed from compound (10) to provide compound (11), the conditions of which will be known by one skilled in the art.Scheme 12

[0090] Scheme 12, step A depicts the SFC purification of compound (11) to give compounds (12) and (13) as diastereomers. In step B, the 3’ alcohol of compound (12) was phosphitylated with 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile in an appropriate solvent such as dichloromethane to provide compound (14). Step C, using the same conditions as step B, provided compound (15) from compound (13).Preparation 1 l-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0091] DIEA (50.1 g, 67.5 mL, 387 mmol) and l-[Chloro-(4-methoxyphenyl)-phenyl- methyl]-4- methoxy-benzene (72.2 g, 213 mmol) were added to a solution of 1- ((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine- 2,4(lH,3H)-dione (50.0 g, 194 mmol) dissolved in DCM (500 mL). The mixture was stirred at ambient temperature for 15 hours. The reaction mixture was quenched with water (500 ml) and the organic layer was removed. The aqueous layer was extracted three times with DCM (200 ml). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / PE, to give the title compound (100 g, 82%) as a yellow solid. ES / MS (m / z): 583.3 (M+23).Preparation 2 l-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0092] Imidazole (216 g, 3.18 mol) and tert-butyldimethylchlorosilane (47.96 g, 52.9 mL, 318 mmol) were added to a solution of l-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2- yl)pyrimidine- 2,4(lH,3H)-dione (100.0 g, 159 mmol) in DMF (500 mL). The mixture was stirred under nitrogen at 50 °C for 15 hours. After cooling to ambient temperature, the mixture was quenched with water (I L). The aqueous layer was extracted three times with EtOAc (800 ml). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / PE, to give the title compound (99 g, 86%) as a yellow solid. ES / MS (m / z): 697.4 (M+23).Preparation 3 l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0093] TFA (59.0 g, 40 ml, 520 mmol) was added to a solution of l-((2R,3R,4R,5R)-5- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (99.0 g, 136 mmol) dissolved in DCM (1000 mL). The mixture was stirred under nitrogen at ambient temperature for 2 hours, cooled to 0 °C in an ice bath, and then quenched by the addition of aqueous sodium bicarbonate (300 mL). The aqueous was then extracted 3 times with DCM (200 mL) and the organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 80% EtOAc / PE, to give the title compound (30 g, 57%) as a yellow solid. ES / MS (m / z): 373.1 (M+l).Preparation 4(2S,3S,4R,5R)-3-((tcrt-butyldimcthylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)- yl)-4-methoxytetrahydrofuran-2-carbaldehyde

[0094] IBX (43.8 g, 156 mmol) was added to a solution of l-((2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)- dione (30.0 g, 78.1 mmol) in EtOAc (400 mL). The mixture was stirred undernitrogen at 80 °C for 5 hours. The reaction mixture was then cooled to ambient temperature, filtered, and concentrated under reduced pressure to give to give the title compound (33 g, 86%) as a pink solid. ES / MS (m / z): 371.1 (M+l).Preparation 5 l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-methoxy-5-vinyltetrahydrofuran-2- yl)pyrimidine-2,4(lH,3H)-dione

[0095] N-Butyllithium (14.6 g, 91.1 mL, 228 mmol, 2.5M in hexane) was added to a solution of methyltriphenylphosphonium bromide (81.4 g, 228 mmol) in THF (600 mL) under nitrogen at -70 °C. The solution was warmed to 0 °C, stirred for 30 minutes, and (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)- yl)-4-methoxytetrahydrofuran-2-carbaldehyde (37.5 g, 75.9 mmol) dissolved in THF (300 mL) was then added. The mixture was then warmed to ambient temperature and stirred under nitrogen for 16 hours. After quenching with saturated ammonium chloride (1200 ml) the mixture was further diluted with water (500 ml). The aqueous was extracted 3 times with EtOAc (1000 ml) and the organic layers were combined, washed with brine (800 ml), dried over sodium sulfate, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 50% EtOAc / PE, to give the title compound (16 g, 56%) as a white solid. ES / MS (m / z): 369.6 (M+l).Preparation 6 l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0096] l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-methoxy-5- vinyltetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (11.1 g, 28.6 mmol) was dissolved in THF (200 mL) under nitrogen and the solution was cooled to 0 °C. 9-BBN in THF (45.2 g, 143 mmol, 0.5M in THF) was added to the solution and it was stirred at 0 °C for 30 minutes. The mixture was then warmed to ambient temperature. After stirring for 16 hours, the solution was cooled to 0 °C and MeOH (12 mL) was added dropwise. When gas evolution had ceased, water (6.0 mL) was added followed by a mixture of sodium hydroxide (2.29 g, 28.6 mL, 57.2 mmol) and hydrogen peroxide (22.7 g, 20.5 ml, 200 mmol). The ice bath was then removed, and the mixture was stirred vigorously at ambient temperature for 2 hours. Aqueous sodium sulfite (400 mL) was then added, and the mixture was extracted 3 times with EtOAc (500 mL). The organic layers were then combined, washed 2 times with brine (200 ml), dried over sodium sulfate, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 50% EtOAc / PE, to give the title compound (8.6 g, 78%) as a colorless oil. ES / MS (m / z): 387.2 (M+l).Preparation 72-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin- l(2H)-yl)-4-methoxytetrahydrofuran-2-yl)acetaldehyde

[0097] l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)- dione (12.0 g, 22.4 mmol) was dissolved in MeCN (150 mL) and IBX (12.5 g, 44.7 mmol) was added. After stirring at 80 °C for 1 hour, the mixture was filtered through a Celite pad and concentrated to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 60% EtOAc / PE, to give the title compound (8.4 g, 86%) as a white solid. ES / MS (m / z): 385.1 (M+l).Preparation 8 l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxypropyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0098] Methylmagnesium bromide (3.49 g, 9.77 mL, 29.3 mmol, 3M in THF) was added to a solution of 2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-4-methoxytetrahydrofuran-2- yl)acetaldehyde (3.20 g, 7.32 mmol) in THF (100 mL) at 0 °C under nitrogen. The solution was stirred at 0 °C for 1 hour. The reaction mixture was quenched by the addition of aqueous saturated ammonium chloride (150 mL) and then diluted with water (100 mL). The aqueous was extracted with EtOAc(200 mL). The organic layer was washed with brine (100 mL), dried over sodium sulfate, filtered, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 20% to 60% EtOAc / PE to give the title compound (1.8 g, 61%) as a colorless oil. ES / MS (m / z): 401.1 (M+l).Preparation 9L((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0099] Silver nitrate (15.2 g, 89.2 mmol), 2,4,6-trimethylpyridine (14.6 g, 15.9 mL, 119 mmol), and 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (20.2 g, 59.7 mmol) were added to a solution of l-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-((RS)-2- hydroxypropyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (5.90 g, 11.9 mmol) in DCM (120 mL) at 0 °C. The ice bath was removed, and the mixture was stirred at ambient temperature for 16 hours under nitrogen. The reaction mixture was quenched by the addition of water (200 mL) and the aqueous was then extracted three times with EtOAc (200 mL). The organic layers were then combined, washed 2 times with brine (200 ml), dried over sodium sulfate, and then reduced to residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 70% EtOAc / PE, to give the title compound (11 g, 74%) as an orange solid. ES / MS (m / z): 725.3 (M+23).Preparation 10 l-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione

[0100] Tetrabutylammonium fluoride (2.50 g, 9.70 mL 9.70 mmol, IM in THF) was added to a solution of l-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4- ((tcrt-butyldimcthylsilyl)oxy)-3-mcthoxytctrahydrofuran-2-yl)pyrimidinc-2,4(lH,3H)-dionc (10.0 g, 9.70 mmol) in THF (100 mL) and the mixture was stirred at ambient temperature under nitrogen. After 30 minutes of stirring, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / PE, to give the title compound (5.0 g, 80%) as a white solid. ES / MS (m / z): 587.2 (M-l).Preparation 11 l-((2R,3R,4R,5R)-5-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione and l-((2R,3R,4R,5R)-5-((R)-2- (bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3-methoxytetrahydrofuran-2- yl)pyrimidine-2,4(lH,3H)-dione

[0101] l-((2R,3R,4R,5R)-5-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy- 3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (5.00 g, 8.15 mmol) was purified by SFC (Condition: CCh-EtOH; Column: Daicel Chiralpak IBN 250 mm X 50 mm X 10 um; Begin B: 50%; End B: 50%; Gradient Time(min): 150 min; Flowrate: 200mL / min.) to give l-((2R,3R,4R,5R)-5-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4- hydroxy-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (first eluting isomer, 1.85 g, 38%) and l-((2R,3R,4R,5R)-5-((R)-2-(bis(4- methoxyphenyl)(phenyl)methoxy)propyl)-4-hydroxy-3-methoxytetrahydrofuran-2- yl)pyrimidine-2,4(lH,3H)-dione (second eluting isomer, 3.01 g, 61%) as white solids.ES / MS (m / z): 587.2 (M-l). Relative stereochemistry was verified by x-ray crystallography.Preparation 12(2R,3R,4R,5R)-2-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-5-(2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0102] l-((2R,3R,4R,5R)-5-((S)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4- hydroxy-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (1.85 g, 3.14 mmol) was dissolved in a mixture of DIEA (1.22 g, 1.64 mL, 9.43 mmol) in DCM (25 mL) and the solution was chilled to 0 °C in an ice bath. 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (1.64 g, 1.54 mL, 6.91 mmol) was then added dropwise and the ice bath was removed. The mixture was stirred at ambient temperature for 1.5 hours and the solvent was then removed under vacuum at 25 °C to leave a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / hexanes (eluents contain 1% TEA) to give the title compound (1.54 g, 62%) as an off-white foam.31P NMR (DMSO) 5 148.90, 148.80; ES / MS (m / z): 789.4 (M+l).Preparation 13(2R,3R,4R,5R)-2-((R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-5-(2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0103] l-((2R,3R,4R,5R)-5-((R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-4- hydroxy-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (3.01 g, 5.11 mmol) was dissolved in a mixture of DIEA (1.98 g, 2.67 mL, 15.3 mmol) in DCM (40 mL) and the solution was chilled to 0 °C in an ice bath. 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (2.66 g, 2.51 mL, 11.2 mmol) was then added dropwise and the ice bath was removed. The mixture was stirred at ambient temperature for 1.5 hours and the solvent was then removed under vacuum at 25 °C to leave a residue. The residue was purified by silica gel flash chromatography, eluting with 0% to 100% EtOAc / hexanes (eluents contain 1% TEA) to give the title compound (3.8 g, 95%) as an off-white foam.31P NMR (DMSO) 5 149.06, 148.99; ES / MS (m / z): 789.4 (M+l).Preparation 14(6,7-Diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)methyl acetate

[0104] To a suspension of (5-acetamido-3,4,6-triacetoxy-tetrahydropyran-2-yl)methyl acetate (25.0 g, 62.9 mmol) in 1,2-DCE (120 mL) was added trimethylsilyl trifluoromethanesulfonate (18.4 mL, 94.3 mmol). The mixture was heated to 50 °C and stirred for 16 hours under nitrogen atmosphere. After this time, the cooled mixture was poured into cold saturated aqueous NaHCCh and extracted three times with DCM. The organic layer was washed successively with saturated aqueous NaHCO, solution and saturated aqueous NaCl solution, dried over MgSCL, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography, eluting with 0-5% MeOH / DCM, to give the title compound as thick, yellow oil (12.7 g, 61%). ES / MS m / z: 330 (M+H).Preparation 15Di-tert-butyl 3,3'-((2-amino-2-((3-(terZ-butoxy)-3-oxopropoxy)methyl)propane-l,3- diy l)bi s (oxy ) )dipropionate

[0105] Tris(hydroxymethyl)aminomethane (7.0 g, 57 mmol) was dissolved in fresh dimethyl sulfoxide (11 mL) and cooled to 15 °C under a nitrogen atmosphere. While stirring, 5N sodium hydroxide (1.2 mL, 6.0 mmol) was added, followed by tert-butyl acrylate (30 mL, 203 mmol) as a slow stream. The mixture was stirred at ambient temperature. After 8 hours, an additional 5N sodium hydroxide (1.2 mL, 6.0 mmol) and tert-butyl acrylate (15 mL, 101 mmol) were added and stirring continued for another 8 hours. The mixture was concentrated under reduced pressure (50 °C and 2 mbar) for 1 hour. The residue was diluted with water and saturated aqueous NaCl solution, then extracted three times with EtOAc. The organic layer was dried over MgSCU, filtered, and concentrated in vacuo to give the title compound as a colorless, viscous oil (24.72 g, 85%). ES / MS m / z: 506 (M+H).Preparation 16Di-terz-butyl 3,3'-((2-(((benzyloxy)cai’bonyl)amino)-2-((3-(?e77-butoxy)-3- oxopropoxy)methyl)propane- 1 ,3-diyl)bis(oxy ))dipropionate

[0106] To di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane- l,3-diyl)bis(oxy))dipropionate (24.7 g, 48.9 mmol) and sodium carbonate (25% aqueous, 180 mL) in DCM (300 mL) was added benzyl chloroformate (22 mL, 151 mmol) as a slow stream. The mixture was stirred at ambient temperature for 4 hours. After this time, the mixture wasdiluted with water, stirred for 5 minutes and the layers separated. The aqueous layer was extracted two times with DCM. The combined organic layer was washed with saturated aqueous NaCl solution, dried over Na2SC>4, filtered and concentrate in vacuo. The crude residue was purified by silica gel flash chromatography, eluting with 0-60% EtOAc in hexanes to give the title compound as thick, colorless oil (19.1 g, 61.1%). ES / MS m / z: 640 (M+H).Preparation 173,3'-((2-(((Benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-l,3- diyl)bis(oxy))dipropionic acid

[0107] Di-terZ-butyl 3, 3'-((2-(( (benzyloxy )carbonyl)amino)-2-((3-(tcrt-butoxy)-3- oxopropoxy)methyl)propane-l,3-diyl)bis(oxy))dipropionate (19.1 g, 29.9 mmol) was stirred in formic acid (100 mL) at ambient temperature for 16 hours. After this time, the mixture was concentrated in vacuo (50 °C and 2 mbar) to give the title compound as a thick oil (15.07 g, 94.2%). ES / MS m / z: 472 (M+H).Preparation 183-(Dibenzylamino)propan-l-ol

[0108] To a mixture of benzyl bromide (16.5 mL, 136 mmol) and 3-amino-l -propanol (5.00 mL, 64.7 mmol) in acetone (200 mL) was added potassium carbonate (45.0 g, 322.3 mmol). The mixture was stirred for 16 hours under nitrogen atmosphere. After this time, the mixture was filtered and the filter cake washed with acetone. The filtrate was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography, eluting with 0-80% EtOAc in hexanes to give the title compound as light, yellow liquid (7.9 g, 48%). ES / MS m / z: 256 (M+H).Preparation 19N,N-Dibcnzyl-3-(3-((tctrahydro-2H-pyran-2-yl)oxy)propoxy)propan-l-aminc

[0109] Tetrabutylammonium sulfate (50 mass% in H2O) (42 mL, 36 mmol) was added to a mixture of 3-(dibenzylamino)propan-l-ol (7.9 g, 31 mmol) and 2-(3- bromopropoxy)tetrahydro-2H-pyran (6.5 mL, 38 mmol) in 5N sodium hydroxide (120 mL). The mixture was stirred at 70 °C for 24 hours. Additional tetrabutylammonium sulfate (50 mass% in H2O) (42 mL, 36 mmol) and 2-(3-bromopropoxy)tetrahydro-2H-pyran (6.5 mL, 38 mmol) were added and stirred for a further 24 hours. After cooling to ambient temperature, the mixture was diluted with saturated aqueous NaCl solution and extracted three times with EtOAc. The organic layer was washed with saturated aqueous NaCl solution, dried overN £12.804. filtered and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography, eluting with 0-50% EtOAc in hexanes to give the title compound as yellow liquid (7.6 g, 62%). ES / MS m / z: 398 (M+H).Preparation 203-(3-((Tetrahydro-2H-pyran-2-yl)oxy)propoxy)propan-l -amine

[0110] Palladium hydroxide on carbon (20% wet) was suspended in MeOH (225 mL) in a Parr shaker. A solution of N,N-dibenzyl-3-(3-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)propan-l- amine in MeOH (225 mL) was added. The shaker was sealed and purged with nitrogen gas, followed by hydrogen gas. The mixture was stirred at ambient temperature under 60 psi of hydrogen gas. After 8 hours, the mixture was filterd over a small pad of diatomaceous earth and the filter cake washed with MeOH. The filtrate was concentrated in vacuo to give the title compound as a yellow liquid (3.95 g, 96%). GC / MS m / z: 218.Preparation 21Benzyl (9,19-dioxo-14-((3-oxo-3-((3-(3-((tetrahydro-2H-pyran-2- yl)oxy)propoxy)propyl)amino)propoxy)methyl)-l,27-bis((tetrahydro-2H-pyran-2-yl)oxy)- 4,12,16,24-tetraoxa-8,20-diazaheptacosan-14-yl)carbamate

[0111] 3,3'-((2-(((Benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-l,3- diyl)bis(oxy))dipropionic acid (2.00 g, 4.20 mmol) and 3-(3-((tetrahydro-2H-pyran-2- yl)oxy)propoxy)propan-l -amine (3.95 g, 18.2 mmol) were dissolved in DMF (50 mL). HBTU (4.90 g, 13.0 mmol) and DIEA (5.00 mL, 27.3 mmol) were added and the mixture stirred at ambient temperature. After 16 hours, the mixture was diluted with ice water and extracted two times with DCM. The organic layer was washed with saturated aqueous NaCl, dried over MgSCL filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography, eluting with 0-20% MeOH in DCM to give the title compound as a thick, yellow oil (3.5 g, 77%). ES / MS m / z: 1092 (M+23).Preparation 22Benzyl (l,27-dihydroxy-14-((3-((3-(3-hydroxypropoxy)propyl)amino)-3- oxopropoxy)methyl)-9,19-dioxo-4,12,16,24-tetraoxa-8,20-diazaheptacosan-14-yl)carbamate

[0112] p-Toluenesulfonic acid monohydrate (0.12 g, 0.68 mmol) was added to a solution of benzyl (9,19-dioxo-14-((3-oxo-3-((3-(3-((tetrahydro-2H-pyran-2- yl)oxy)propoxy)propyl)amino)propoxy)methyl)-l,27-bis((tetrahydro-2H-pyran-2-yl)oxy)- 4,12,16,24-tetraoxa-8,20-diazaheptacosan-14-yl)carbamate (3.5 g, 3.3 mmol) in MeOH (50 mL). The mixture was stirred at 60 °C for 30 minutes, cooled to ambient temperature then concentrated in vacuo. The residue was diluted with saturated aqueous NaHCOi solution and extracted three times with chloroform. The organic layer was washed successively with saturated aqueous NaHCOs and saturated aqueous NaCl solution, dried over MgSO4, filteredand concentrated in vacuo to give the title compound as a thick, yellow oil (2.57 g, 96%).ES / MS m / z: 409 ([M+2H] / 2).Preparation 23[5-Acetamido-6-[3-[3-[3-[3-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]-2-[[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]methyl]-2-(benzyloxycarbonylamino)propoxy]propanoylamino]propoxy]propoxy]-3,4-diacetoxy- tetrahydropyran-2-yl]methyl acetate

[0113] To a mixture of benzyl (l,27-dihydroxy-14-((3-((3-(3- hydroxypropoxy)propyl)amino)-3-oxopropoxy)methyl)-9,19-dioxo-4,12,16,24-tetraoxa-8,20- diazaheptacosan-14-yl)carbamate (2.5 g, 3.1 mmol) in 1,2-dichloroethane (30 mL) was added 4 A molecular sieves (3 g), (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]oxazol-5-yl)methyl acetate (0.3M in 1,2-dichloroethane, 30 mL, 9 mmol) and trimethylsilyl trifluoromethanesulfonate (0.2 mL, 1.02 mmol). The mixture was stirred for 16 hours at ambient temperature under nitrogen atmosphere. After this time, the mixture was diluted with saturated aqueous NaHCCL solution and extracted three times with chloroform. The organic layer was dried over MgSCL. filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography, eluting with 0-20% MeOH in DCM, to give the title compound as colorless foam (850 mg, 15%). ES / MS m / z: 903 ([M+2H] / 2).Preparation 24 [5-Acetamido-6-[3-[3-[3-[3-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]-2-[[3-[3-[3- [3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propanoylamino]propoxy]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate

[0114] [5-Acetamido-6-[3-[3-[3-[3-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]-2-[[3-[3-[3- [3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]methyl]-2- (benzyloxycarbonylamino)propoxy]propanoylamino]propoxy]propoxy]-3,4-diacetoxy- tetrahydropyran-2-yl]methyl acetate (0.85 g, 0.47 mmol) was combined with palladium on carbon (50% wet) (0.1 g, 0.047 mmol) in MeOH (10 mL). Two drops of glacial acetic acid was added. The mixture was stirred under 1 atmosphere of hydrogen gas. After 2 hours, the mixture was filtered over a small pad of diatomaceous earth and the cake washed with MeOH. The filtrate was concentrated in vacuo to give the title compound as a colorless foam (726 mg, 92%). ES / MS m / z: 836 ([M+2H] / 2).Preparation 25Benzyl 12-[[2-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo- propoxy]methyl]ethyl]amino]-12-oxo-dodecanoate

[0115] To 12-benzyloxy-12-oxo-dodecanoic acid (0.100 g, 0.312 mmol) in DMF (2 mL) was added HBTU (0.121 g, 0.313 mmol) and D1EA (0.18 mL, 0.982 mmol). The mixture was stirred for 5 minutes then [5-acetamido-6-[3-[3-[3-[3-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]-2-[[3-[3-[3- [3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propanoylamino]propoxy]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (0.400 g, 0.239 mmol) in DMF (2 mL) was added. The mixture was stirred at ambient temperature for 2 hours. After this time, the mixture was diluted with water and extracted three times with chloroform. The organic layer was washed successively with saturated aqueous NaHCCL and saturated aqueous NaCl solution, dried over MgSCU, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography, eluting with 0-30% MeOH in DCM, to give the title compound as a colorless foam (176 mg, 37%). ES / MS m / z: 988 ([M+2H] / 2).Preparation 2612-[[2-[3-[3-[3-[3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]methyl]ethyl]amino]- 12-oxo-dodecanoic acid

[0116] Palladium on carbon (50% wet) (0.0200 g, 0.00939 mmol) was added to a solution of benzyl 12-[[2-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-ylJoxypropoxyJpropylamino]-3-oxo- propoxy]methyl]ethyl] amino] -12-oxo-dodecanoate (0.176 g, 0.0892 mmol) in MeOH (3 mL). The mixture was stirred under 1 atmosphere of hydrogen gas. After 18 hours, the mixture was filtered through a short pad of diatomaceous earth and the filter cake rinsed with MeOH. The filtrate was concentrated in vacuo to give the title compound as a colorless foam (158 mg, 94%). ES / MS m / z: 943 ([M+2H] / 2).Preparation 27N-(((9H-Fhioren-9-yl)methoxy)carbonyl)-O-(bis(4-methoxyphenyl)(phenyl)methyl)-L-serine

[0117] To a stirring solution of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-hydroxy- propanoic acid (40 g, 0.122 mol) in dry DCM (400 mL) was added DIEA (64 mL, 0.366 mol)at 0 °C under inert atmosphere. To this, a solution of DMTC1 (49.6 g, 0.146 mol) in DCM (200 mL) was added slowly. The resulting mixture was brought to ambient temperature and stirred for 16 hours. After this time, the reaction mixture was diluted with water (12.5 vol) and extracted with DCM (25 vol). The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated in vacuo. The crude obtained was washed with 10% EtOAc / hexane (12.5 vol) and dried under vacuum to give the title compound as a pale brown solid (62 g, crude). This material was taken to next step without any further purification. TLC: 5% MeOH / DCM (Rf. 0.5) UV, 254 nM.Preparation 28(9H-Fluoren-9-yl)methyl (S)-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-l-(4-(hydroxymethyl)piperidin- 1 -yl)- 1 -oxopropan-2-y l)carbamate

[0118] To a stirring solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(bis(4- methoxyphenyl)(phenyl)methyl)-L-serine (62 g, 0.103 mol) in DCM (750 mL) was added slowly HBTU (78.3 g, 0.206 mol), HOBt (27.9 g, 0.206 mol), and piperidin-4-yl methanol (15.4 g, 0.134 mol) followed by TMP (15 mL, 0.113 mol) at 0 °C under inert atmosphere. The resulting mixture was brought to ambient temperature and stirred for 4 hours. After this time, the reaction mixture was diluted with water (8 vol) and extracted with DCM (15 vol). The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography, eluting with 20-40% EtOAc / hexane and 1% MeOH / DCM, to give the title compound (40 g, 52% over two steps).*H NMR (DMSO-d6) 57.88 (br d, J = 7.5 Hz, 2H), 7.79 - 7.59 (m, 3H), 7.45 - 7.12 (m, 13H), 6.92 - 6.76 (m, 4H), 4.79 - 4.44 (m, 2H), 4.32 (br d, 7 = 11.4 Hz, 2H), 4.20 (br s, 2H), 3.71 (s, 6H), 3.21 (br s, 4H), 2.99 - 2.79 (m, 1H), 2.69 ( br s, 2H), 1.81 - 1.43 (m, 3H), 1.08 - 0.73 (m, 2H).Preparation 29(S)-2-Amino-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-l-(4-(hydroxymethyl)piperidin-l- yl)propan-l-one

[0119] A solution of 20% piperidine in DMF (400 mL) was added slowly to (9H-fluoren-9- yl)methyl (S)-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)- l-(4-(hydroxymethyl)piperidin- 1- yl)-l-oxopropan-2-yl)carbamate (40 g, 0.055 mol) at 0 °C under inert atmosphere. The resulting reaction mixture was stirred at ambient temperature for 1 hour. After this time, the mixture was diluted with water (15 vol) and extracted with EtOAc (30 vol). The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography, eluting with 1-8% MeOH / DCM, to give the title compound as an off-white solid (13 g, 47%). ES / MS m / z 1009.5 (2M+H).Preparation 30N~1 — { l,27-Bis[(2-Acetamido-3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]-14-({3-[(3-{3-[(2-acetamido-3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]propoxy}propyl)amino]-3-oxopropoxy }methyl)-9,19-dioxo-4, 12, 16,24-tetraoxa-8,20-diazaheptacosan-14-yl}-n~ 12 — {(2s)-3-[bis(4-methoxyphenyl)(phenyl)methoxy]-l-[4-(hydroxymethyl)piperidin-l-yl]-l- oxopropan-2-yl} dodecanediamide

[0120] 12-[[2-[3-[3-[3-[3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo- propoxy]methyl]ethyl] amino] -12-oxo-dodecanoic acid (500 mg, 266 pmol) was added to a solution of (Z)-2-( 1 H-benzo[d] [ 1 ,2,3]triazol- 1 -yl)- 1 , 1 ,3-trimethylisouronium hexafluorophosphate(V) (107 mg, 292 pmol), 1 -hydroxy- IH-benzotriazole (39.5 mg, 292 pmol), and DIEA (228 pL, 1.33 mmol) dissolved in DMF (6 ml). The solution was stirred at ambient temperature for 15 minutes, (S)-2-amino-3-(bis(4-methoxyphenyl)(phenyl)methoxy)- l-(4-(hydroxymethyl)piperidin-l-yl)propan-l-one (147 mg, 292 pmol) was then added, and stirring at ambient temperature was continued for 16 hours. The mixture was diluted with EtOAc and the organic was washed with saturated aqueous NaCl, dried over sodium sulfate, and concentrated in vacuo to give an oil. The oil was purified by silica gel flash chromatography, using 0-20% MeOH / EtOAc (w / 1% triethylamine added), to give the title compound as an off-white foam (418 mg, 66%). ES / MS m / z: 1183.8 ([M-2H] / 2).Preparation 314-((l-((S)-30-(((2S,3S,4S,5S,6S)-3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17,17-bis((3-((3-(3-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propoxy)propyl)amino)-3- oxopropoxy)methyl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4,15,22-trioxo- 19,27-dioxa-3,16,23-triazatriacontanoyl)piperidin-4-yl)methoxy)-4-oxobutanoic acid

[0121] Succinic anhydride (118 mg, 1.18 mmol) was added to a solution of N~l~-{ 1,27- bis[(2-acetamido-3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]-14-({3-[(3-{3-[(2- acetamido-3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]propoxy}propyl)amino]-3- oxopropoxy } methyl)-9, 19-dioxo-4, 12, 16,24-tetraoxa- 8 ,20-diazaheptacosan- 14-yl] -n~ 12 — {(2s)-3-[bis(4-methoxyphenyl)(phenyl)methoxy]-l-[4-(hydroxymethyl)piperidin-l-yl]-l- oxopropan-2-yl]dodecanediamide (1.40 g, 591 pmol), 4-dimethylaminopyridine (217 mg, 1.77 mmol), and triethylamine (412 p.L, 2.95 mmol) dissolved in DCM (20 ml). The mixture was stirred at ambient temperature for 16 hours then purified directly by silica gel flash chromatography, eluting with 0-10% MeOH / DCM (w / 1% triethylamine added), to give the title compound as an off-white foam (1.23 g, 84%). ES / MS m / z: 1234.0 ([M-2H] / 2).Preparation 32Resin loading

[0122] In a resin loading cartridge, 4-((l-((S)-30-(((2S,3S,4S,5S,6S)-3-acetamido-4,5- diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17,17-bis((3-((3-(3- (((2S,3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)propoxy)propyl)amino)-3-oxopropoxy)methyl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4,15,22-trioxo-19,27-dioxa-3,16,23- triazatriacontanoyl)piperidin-4-yl)methoxy)-4-oxobutanoic acid (1.23 g, 499 pmol) was added to a solution of 2-(lH-benzo[d][l,2,3]triazol-l-yl)-l,l,3,3-tetramethylisouronium hexafluorophosphate(V) (391 mg, 1.030 mmol) and DIEA (264 pL, 1.52 mmol) in acetonitrile (50 ml) and the mixture was shaken at ambient temperature for 15 minutes. After this time, native amino LCAA 500 A controlled-pore glass (CPG) resin (3.91 g, 123 pmol / g loading, purchased from ChemGenes) was added and the mixture was shaken at ambient temperature for 16 hours. The cartridge was drained by suction filtration and the CPG was washed by shaking with DCM (50 mL) for 10 minutes. The cartridge was drained and the washing and draining procedure was repeated with 10% MeOH / DCM (50 mL) and Et O (50 mL). After draining, a solution of CAP A (50 ml) was added to the cartridge and shaken at ambient temperature for 2 hours. The cartridge was then drained and the washing and draining procedure above was repeated using DCM (50 mL), 10% MeOH / DCM (50 mL) and diethyl ether (50 mL). The cartridge was drained one final time, and the resin dried under vacuum for 30 minutes. The resin loading was determined using a standard trityl assay. The resin loading was calculated to be 64.1 pmol / g.Preparation 33(2,5-Dioxopyrrolidin-l-yl) 12-[[2-[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]methyl]ethyl]amino]-12-oxo-dodecanoate

[0123] 12-[[2-[3-[3-[3-[3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropoxy]propylamino]-3-oxo- propoxy]methyl]ethyl] amino] -12-oxo-dodecanoic acid (158 mg, 0.0839 mmol) and N- hydroxysuccinimide (15 mg, 0.128 mmol) were dissolved in DCM (1 mL). EDCI HC1 (25 mg, 0.128 mmol) was added and the resulting mixture stirred at ambient temperature for 6 hours. The mixture was loaded directly on a column and purified by silica gel flash chromatography, eluting with 0-40% MeOH in DCM. The desired fractions were concentrated in vacuo. The residue was dissolved in DCM and filtered through glass wool to remove insoluble particulates. The filtrate was concentrated in vacuo to give the title compound as a colorless foam (114 mg, 69%). ES / MS m / z: 990 ([M+2H] / 2).Preparation 3419-[(2-Acetamido-3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]-n-(3-{3-[(2-acetamido- 3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]propoxy}propyl)-6-({3-[(3-{3-[(2-acetamido- 3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]propoxy}propyl)amino]-3-oxopropoxy }methyl)-6-{ 12-[(2s,4r)-2-{ [bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4- hydroxypyrrolidin- 1 -yl] - 12-oxododecanamido } - 11 -oxo-4, 8 , 16-trioxa- 12-azanonadecan- 1 - amide

[0124] 12-[[2-[3-[3-[3-[3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2- yl]oxypropoxy]propylamino]-3-oxo-propoxy]-l,l-bis[[3-[3-[3-[3-acetamido-4,5-diacetoxy- 6-(acetoxymethyl)tetrahydropyran-2-ylJoxypropoxyJpropylamino]-3-oxo- propoxy]methyl]ethyl] amino] -12-oxo-dodecanoic acid (500 mg, 266 pmol) was added to a solution of (Z)-2-( lH-benzo[d] [ l,2,3]triazol- 1-yl)- 1 , 1 ,3-trimethylisouronium hexafluorophosphate(V) (1 11 mg, 292 pmol), 1 -hydroxy- 1 H-benzotriazole (39.5 mg, 292 pmol), and diisopropylethylamine (172 mg, 228 pL, 1.33 mmol) dissolved in DMF (6 mL). The solution was stirred at ambient temperature for 15 minutes, (3R,5S)-5-((bis(4- methoxyphenyl) (phenyl)methoxy)methyl)pyrrolidin-3-ol (123 mg, 292 pmol) was then added, and stirring at ambient temperature was continued for 16 hours. The mixture was diluted with EtOAc and the organic was washed with saturated aqueous NaCl, dried over sodium sulfate, and concentrated in vacuo to give an orange foam. The foam was purified by silica gel flash chromatography, using 0-20% MeOH / EtOAc (w / 1% triethylamine added), to give the title compound as tan foam (313 mg, 52%). ES / MS (m / z): 11414 (M-2H / 2).Preparation 354-(((3R,5S)-l-(l-(((2R,3R,4R,5R,6R)-3-Acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-14,14-bis((3-((3-(3-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)propoxy)propyl)amino)-3-oxopropoxy)methyl)-9,16-dioxo-4,12-dioxa-8,15- diazaheptacosan-27-oyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3- yl)oxy)-4-oxobutanoic acid

[0125] Succinic anhydride (27.4 mg, 274 pmol) was added to a solution of 19-[(2-acetamido- 3,4,6-tri-o-acetyl-2-deoxyhexopyranosyl)oxy]-n-(3-{3-[(2-acetamido-3,4,6-tri-o-acetyl-2- deoxyhexopyranosyl)oxy]propoxy}propyl)-6-({3-[(3-{3-[(2-acetamido-3,4,6-tri-o-acetyl-2- deoxyhexopyranosyl)oxy]propoxy}propyl)amino]-3-oxopropoxy]methyl)-6-{ 12-[(2s,4r)-2- { [bis(4-methoxyphenyl)(phenyl)methoxy]methyl } -4-hydroxypyrrolidin- 1 -yl] - 12- oxododecanamido}-l l-oxo-4,8,16-trioxa-12-azanonadecan-l-amide (313 mg, 137 pmol), 4- dimethylaminopyridine (50.1 mg, 410 pmol), and triethylamine (13.8 mg, 19.1 pL, 137 pmol) dissolved in DCM (5 mL) and the mixture was stirred at ambient temperature for 16 hours. The mixture was purified directly by silica gel flash chromatography, using 0- 10% MeOH / DCM (w / 1% triethylamine added), to give the title compound as an off-white foam (274 mg, 84%). ES / MS (m / z): 1 191 .2 (M-2H / 2).

[0126] In a resin loading cartridge, 2-(lH-benzo[d][l,2,3]triazol-l-yl)-l,l,3,3- tetramethylisouronium hexafluorophosphate(V) (87.3 mg, 230 pmol) and N,N- diisopropylethylamine (44.6 mg, 60.1 pL, 345 pmol) were added to a solution of 4- (((3R,5S)-l-(l-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-14,14-bis((3-((3-(3-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)propoxy)propyl)amino)-3-oxopropoxy)methyl)-9,16-dioxo-4,12-dioxa-8,15- diazaheptacosan-27-oyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3- yl)oxy)-4-oxobutanoic acid (274 mg, 115 pmol) dissolved in a mixture of acetonitrile (10 mL) and DMF (1 mL). The mixture was shaken at ambient temperature for 15 minutes. After this time, Native amino LCAA 500A CPG (1.15 g, 123 pmol / g loading, purchased from ChemGenes) was added and the mixture was shaken at ambient temperature for 16 hours. The cartridge was drained by suction filtration and the CPG was washed by shaking with DCM (10 mL) for 10 minutes. The cartridge was drained and the washing and draining procedure was repeated with 10% MeOH / DCM (10 mL) and EtoO (10 mL). After draining, a solution of CAP A (10 mL) was added to the cartridge, and it was shaken at ambient temperature for 2 hours. The cartridge was then drained and the washing and draining procedure above was repeated using DCM (10 mL), 10% MeOH / DCM (10 mL) and diethylether (10 mL). The cartridge was drained one final time, and the CPG was dried under vacuum for 45 minutes. The resin loading was determined using a standard trityl assay. The resin loading was calculated to be 42.9 pmol / g.EXAMPLE 1Synthesis of oligonucleotides

[0127] The sense strand and antisense strand of an RNAi agent including at least one PrON can be synthesized using any nucleic acid polymerization methods known in the ait, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-l,2,4- dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.

[0128] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a duplex.

[0129] In other embodiments disclosed herein are RNAi agents having a formula of R-L-D, wherein R is a double stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the antisense strand is complementary to target mRNA, wherein L is a bond or a linker, and wherein D is a delivery moiety of Formula II:Formula II.

[0130] In some embodiments, the RNAi agent disclosed herein comprises a linker. In further embodiments, R is conjugated to Formula II via a linker. In other further embodiments R is conjugated to Formula II via a linker. In further embodiments, the linker comprises a linker of Formula III having connection points A and B or the linker comprises Formula IV having connection points X and Y, and wherein:Formula IV ; a. the RNAi agent comprises Formula II conjugated to Formula III at connection point A and Formula III is conjugated to a phosphate group at connection point B, and the phosphate group is conjugated to R; or b. the RNAi agent comprises Formula II conjugated to Formula IV at connection point X and Formula IV is conjugated to a phosphate group at connection point Y, and the phosphate group is further conjugated to R.

[0131] In an embodiment in which the RNAi agent of Formula II includes a linker, and the linker is of Formula IV, -L-D has the structure of Formula V :Formula V, wherein dsRNA R is connected to Formula V at connection point Y via a phosphate or phosphate analog.

[0132] The delivery moiety of Formula II may be made by synthetic steps and schemes as described in United States Patent Application No. 18 / 573,794, filed December 22, 2023, or International Patent Publication No. WO2022 / 271806, each of which is incorporated herein by reference in its entirety.

[0133] Briefly, for the synthesis of GalNAc-conjugated sense strands, a sense strand containing an appropriate conjugation handle, may first be synthesized using standard phosphoramiditc chemistry. A stock solution of the desired GalNAc ligand-NHS ester (10 mmol / L in acetonitrile; 1 eq) is prepared. Borate buffer (10% v / v; 20x) is added to oligonucleotide sense strand in an Eppendorf tube, then GalNAc ligand (5 eq) is added. The mixture is shaken at ambient temperature for 16 hours. After this time, the mixture is transferred to a 15 mL falcon tube, ammonium hydroxide (28 mass%) is added, and the mixture is shaken at ambient temperature for 2 hours. The ammonia is then removed in vacuo. The residue is purified by ion-exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15%MeCN / 20mM NaH2PO4, IM NaBr; 35-55%B over 5 CV at 8 mL / min, column temperature 60 °C. The desired fractions are pooled anddesalted by spin-filtration using an Eppendorf centrifuge or desalting column. After desalting, the material is recovered and OD and volume are measured to obtain concentration.

[0134] Alternatively, and particularly in the case of a delivery moiety including a linker of Formula IV, oligo synthesis may be conducted on a MerMade™ 12 instrument using standard phosphoramidite chemistry and GalNAc functionalized CPG. Sense strands are synthesized from the prefunctionalized GalNAc solid support and antisense strands are synthesized using standard support preloaded with the first nucleotide of the oligo sequence. Antisense strands are prepared using CPG with a universal support or linker. Oligos are cleaved and deprotected by shaking the CPG in ammonium hydroxide solution (28% by mass) at 40 degrees for approximately 16 hours, filtered to remove CPG, and then purified by ion exchange chromatography using conditions described above. Such deprotection likewise converts the OAc precursor groups of the galactose moieties to hydroxyl groups. Desalting, annealing, and endotoxin testing are conducted.

[0135] To generate the siRNA duplexes of a sense and antisense strand, the following procedures may be performed. To a tube (such as a Falcon tube) containing oligonucleotide sense strand-GalNAc conjugate, the corresponding antisense oligonucleotide (1 eq) is added and vortcxcd for 10 seconds before spin-filtering through 100K MWCO Amicon filter unit to remove particulates. The filtrate is recovered and concentrated in vacuo on a Genevac evaporator. The residue is reconstituted in lx PBS, filtered through 0.2 p filter, and OD and volume are measured to obtain concentration.

[0136] In further embodiments of the RNAi agents disclosed herein, the sense strand and antisense strand each independently comprise one or more modified intemucleotide linkages, and each modified internucleotide linkage is a phosphorothioate linkage. In further embodiments, the sense strand and antisense strand each independently comprise four phosphorothioate linkages. In still further embodiments, the two terminal nucleotides at each of the 5’ and 3’ ends of each of the sense and antisense strand are phosphorothioate linkages.

[0137] In certain embodiments, the delivery moiety may instead be a compound comprising Formula VI:Formula VI wherein Formula VI may be conjugated to an oligonucleotide directly or via a linker, wherein the linker or oligonucleotide is conjugated to the delivery moiety at connection point E.

[0138] In an embodiment, the linker comprises a linker of Formula VII having connection points A and B; or of Formula VIII having connection points C and D; or of Formula IX having connection points E and F:Formula VIII;Formula IX; wherein connection point A or connection point C or connection point E are conjugated to Formula VI.

[0139] In another aspect, the delivery moiety may comprise a compound of Formula X:Formula X.

[0140] In another embodiment, the delivery moiety may comprise a compound of FormulaXI:Formula XI.

[0141] In another embodiment, the delivery moiety may comprise a compound of FormulaXII:

[0142] In another embodiment, the delivery moiety may comprise a compound of FormulaXIII:Formula XIII.

[0143] In another embodiment, the delivery moiety may comprise a compound of FormulaXIV:Formula XIV.

[0144] In another embodiment, the delivery moiety may comprise a compound of FormulaXV:Formula XV.

[0145] In another embodiment, the delivery moiety may comprise a compound of FormulaXVI:Formula XVI.

[0146] In another embodiment, the delivery moiety may comprise a compound of FormulaXVII:Formula XVII.

[0147] In another embodiment, the delivery moiety may comprise a compound of FormulaXVIII:Formula XVIII.

[0148] It will be appreciated that any of the delivery moieties disclosed herein may be conjugated to an oligonucleotide directly, via any linker disclosed herein, by any combination of linkers disclosed herein, and by other linkers.

[0149] For example, the delivery moiety D of Formula Xa may be conjugated to the linker of Formula IX, to yield a linker-delivery moiety L-D of Formula XIX:Formula XIX.

[0150] In some embodiments, the linker-delivery moiety L-D of Formula XIX may be conjugated to a nucleic acid, such as via a phosphate group at the 3’ end of the nucleic acid, at connection point F. For example the oxygen at connecting point

[0151] In another embodiment, the delivery moiety of Formula VI may be conjugated to the linker of Formula VIII, to yield a linker-delivery moiety L-D of Formula XX:Formula XX.

[0152] In some embodiments, the linker-delivery moiety L-D of Formula XX may be conjugated to a nucleic acid, such as via a phosphate group at the 3’ end of the nucleic acid, at connection point F.

[0153] It will be appreciated that the linkers as shown herein may be depicted in different ways, and will form covalent bonds with the delivery moiety and / or the nucleic acid of the RNAi agent in conventional ways. For example, when a linker is depicted with an oxygen atom at the connection point, and is to be conjugated to a phosphate group of a nucleic acid, it will be understood that the oxygen of the linker will take the place of an oxygen atom of the nucleic acid. When a linker is not shown with an oxygen atom at the connection point, an oxygen atom of the nucleic acid may in some embodiments covalently bind the linker at the depicted position, and so forth.

[0154] 2-propyl nucleotides (PrON) were incorporated into oligonucleotides using standard phosphoramidite chemistry and standard synthetic methods. The compounds of Preparations 12 and 13 above were used in CPG methods described herein.

[0155] In certain embodiments, the 5’ nucleotide of the antisense strand comprises a phosphate group or a phosphate analog.

[0156] In some embodiments, at least one of the sense strand and the antisense strand can include at least one abasic moiety of the formula:wherein “5”’ and “3”’ indicate the direction of the sequences.

[0157] In some embodiments, at least one of the sense strand and the antisense strand can include at least one inverted abasic (iAb) moiety of the formula:5’ / rZ°3' wherein “5”’ and “3”’ indicate the direction of the sequences.

[0158] An endotoxin test may be performed using a Limulus amebocyte lysate on an EndosafeO-nexgen PTS instrument.Example 2: Conjugation of the delivery moiety

[0159] For the synthesis of GalNAc-conjugated sense strands, a sense strand with a 3’ C6- NH2 functional group was first synthesized using standard phosphoramidite chemistry. A stock solution of GalNAc ligand-NHS ester (10 mmol / L in acetonitrile; 1 eq) was prepared. Borate buffer (10% v / v; 20x) was added to oligonucleotide C6-NH2 sense strand in an Eppendorf tube, then GalNAc ligand (5 eq) was added. The mixture was shaken at ambient temperature for 16 hours. After this time, the mixture was transferred to a 15 mL falcon tube, ammonium hydroxide (28 mass%) was added, and the mixture was shaken at ambient temperature for 2 hours. The ammonia was then removed in vacuo. The residue was purified by ion-exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15%MeCN / 20mM NaH2PO4, IM NaBr; 35-55%B over 5 CV at 8 mL / min, column temperature 60 °C. The desired fractions were pooled and desalted by spinfiltration using an Eppendorf centrifuge or desalting column. After desalting, the material was recovered and OD and volume were measured to obtain concentration.

[0160] Alternatively, conjugation to the 3’ position of the sense strand through immobilizing the GalNAc ligand on microporous polystyrene resin or controlled pore glass and synthesized using established solid phase oligonucleotide synthesis methods with 5’-CE B-cyanoethyl) phosphoramidites.

[0161] Alternatively, the GalNAc ligand was converted to a suitable phosphoramidite and delivered to the 5’ position of the sense strand using standard phosphoramidite chemistry.EXAMPLE 3General procedure for oligo synthesis using GalNAc-functionalized CPG

[0162] Oligo synthesis was conducted on a MerMade™ 12 instrument using phosphoramidite chemistry. Sense strands were synthesized from the prefunctionalized GalNAc solid support and antisense strands were synthesized using standard support preloaded with the first nucleotide of the oligo sequence. Oligos were cleaved and deprotected using concentrated ammonium hydroxide solution (28% by mass) and purifiedby ion exchange chromatography using conditions described above. Desalting, annealing, and endotoxin testing were conducted.

[0163] The sequence of antisense oligonucleotides were designed using 15 to 50 nucleotides of the antisense strands described herein, including those in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, and Table 5.

[0164] Exemplary antisense strand sequences of 23 nucleotides in length are shown in Table 2A, Table 2B, Table 3A, Table 3B, Table 4, and Table 5 below, which may be optionally further modified and synthesized and incorporated into the RNAi agents, as described herein.EXAMPLE 4Incorporation of PrON into nucleic acids

[0165] As mentioned previously, PrON can be incorporated into oligonucleotides of the present disclosure using standard phosphoramidite chemistry and conventional nucleic acid synthesis methods.

[0166] A nucleic acid may incorporate as many PrON as there are nucleotides in the molecule. In one aspect, an oligonucleotide incorporates 1-5 PrON, inclusive, or 1 PrON, or 2 PrONs, or 3 PrONs, or 4 PrONs, or 5 PrONs. The PrONs may be consecutive, that is in adjacent positions and bonded to one another, or they may be spaced out throughout the length of the oligonucleotide. The PrON may be at the 5’ end of an oligonucleotide, or at the 3’ end of the oligonucleotide, or at any internal position. In one example, for an RNAi agent which is a duplex RNA including a sense strand and an antisense strand, the PrON may be at the 3’ end of the antisense strand, such as in an overhang (that is, not within a duplex region of the dsRNA molecule).

[0167] When a PrON is incorporated into an oligonucleotide, it may be incorporated with a random stereochemistry (that is, R or S) by using a racemic mixture of precursor compounds such as phosphoramidites, or it may done in with specific stereochemistry, such as by using the enantiopure phosphoramidite having the S intemucleotide linkage of Preparation 12 above, or the R internucleotide linkage of Preparation 13. In some instances, multiple PrONs may be included in a single oligonucleotide, such as two consecutive positions at the 3’ endof an oligonucleotide which may serve as an antisense strand of a double- stranded RNAi agent. In these cases, the nucleobase may be any nucleobase listed herein, such as, for example, uracil. In some embodiments, the oligonucleotide in which PrON is incorporated may have a 5’ phosphate, or a 5’ hydroxyl, or a 5’ vinyl phosphate group.

[0168] The sequences listed in tables 2A, 2B, 3 A, and 3B may be modified to incorporate PrON at any position. In specific embodiments, the antisense sequences in these tables may be modified in order to incorporate one or more PrON. In a specific embodiment, the 23- nucleotide antisense strands listed in these tables (SEQ ID NO: 6-10 of Table 2A, SEQ ID NO: 11-15 of Table 2B, SEQ ID NO: 113-209 of Table 3A, and SEQ ID NO: 210-306 of Table 3B) may have their two 3’ nucleotides substituted for PrON with the same or different nucleobases. For instance, in SEQ ID NO: 6 of Table 2A, the guanine and uracil at the 3’ end can each be substituted by PrON bearing uracil as a nucleobase at positions 22 and 23. The PrON can have any stereochemical configuration, such as:(a) R-PrON at position 22 and R-PrON at position 23; or(b) R-PrON at position 22 and S-PrON at position 23; or(c) S-PrON at position 22 and R-PrON at position 23; or(d) S-PrON at position 22 and S-PrON at position 23.The skilled artisan will readily appreciate that other modifications can be made along with incorporation of PrON in such molecules.EXAMPLE 5In vitro knockdown of human Ang8 in Hep3B cells with GalNAc-conjugated Ang8 siRNA by transfection

[0169] For Hep3B (ATCC, Part #: HB-8064) cells, transfection was carried out by adding 24.7 pL of Opti-MEM™ (Gibco, Part #: 31985062) plus 0.3 pL of Lipofectamine™ RNAiMAX (Life Technologies, Part #: 13778-150) per well to 25 pL of each siRNA duplex to an individual well in a 96-well plate (Costar, Part #3596). RNAi duplexes were conjugated with Formula V as shown above on the 3’ end of the sense strand. The mixture was then incubated at room temperature for 20 minutes. Fifty microliters of complete growthmedia without antibiotic containing Hep3B at 400,000 cells / mL were then added to the siRNA mixture. 10 points concentration related curves (CRC) was done at siRNA duplex final concentration starts at 20nM, then 1:5 serial dilution. Cells were incubated for 24 hours prior to qPCR.

[0170] Treated cells were washed with cold IxPBS and lysed directly into the 96 well cell plate using TaqMan™ Fast Advanced Cell-to-CT™ kit (Life Technologies, Part #: AM1729). cDNA was synthesized using the following steps in a thermocycler: 37°C for 30 minutes, 95°C for 5 minutes, and 4°C hold. Polymerase Chain Reaction (PCR) using the following cycles temperatures and times: 50°C for 2 minutes, 95°C for 20 seconds, 40 cycles of 95°C for 1 seconds and 60°C for 20 seconds.

[0171] The human Ang8 levels were normalized to human RPLP0 (Life Technologies) and represented the relative knockdown of human Ang8 mRNA expression as compared to vehicle-treated control cells.Table 1: Hep3B / RNAiMAXTable 2A: Modified ANGPTL8 Sequences for RNAi AgentsTable 2B: Modified ANGPTL8 Sequences for RNAi AgentsTable 3A: Modified sense and antisense strands for ANGPTL8 RNAi agentsP or [Phos] indicates a 5’ phosphate m indicates 2’0-methyl modified ribose on the listed nucleotide f indicates 2’F modified ribose on the listed nucleotide* indicates a phosphorothioate bond (in place of a phosphodiester bond)Table 3B: Modified sense and antisense strands for the ANGPTL8 RNAi agentsm indicates 2’0-methyl modified ribose on the listed nucleotide f indicates 2’F modified ribose on the listed nucleotide* indicates a phosphorothioate bond (in place of a phosphodiester bond)Table 4: Sense and antisense strands for ANGPTL8 RNAi agentsTable 5Modified sense and antisense strands with PrON or exNA for the ANGPTL8RNAi Agents hereinAbbreviations - “m” indicates 2’-0Me; “f” indicates 2’-fluoro; indicates phosphorothioate linkage; “S” means the sense strand; “AS” means the antisense strand; rPrON indicates PrON with R stereocenter in the intemucleotide linkage; sPrON indicates PrON with S stereocenter in the intemucleotide linkage; 5ex indicates exNA. human ANGPTL8 transcript NM_018687.7 SEQ ID 511: ataccttaga ccctcagtca tgccagtgcc tgctctgtgc ctgctctggg ccctggcaat ggtgacccgg cctgcctcag cggcccccat gggcggccca gaactggcac agcatgagga gctgaccctg ctcttccatg ggaccctgca gctgggccag gccctcaacg gtgtgtacag gaccacggag ggacggctga caaaggccag gaacagcctg ggtctctatg gccgcacaat agaactcctg gggcaggagg tcagccgggg ccgggatgca gcccaggaac ttcgggcaag cctgttggag actcagatgg aggaggatat tctgcagctg caggcagagg ccacagctga ggtgctgggg gaggtggccc aggcacagaa ggtgctacgg gacagcgtgc agcggctaga agtccagctg aggagcgcct ggctgggccc tgcctaccga gaatttgagg tcttaaaggc tcacgctgac aagcagagcc acatcctatg ggccctcaca ggccacgtgc agcggcagag gcgggagatg gtggcacagc agcatcggct gcgacagatc caggagagac tccacacagc ggcgctccca gcctgaatct gcctggatgg aactgaggac caatcatgct gcaaggaaca cttccacgcc ccgtgaggcc cctgtgcagg gaggagctgc ctgttcactg ggatcagcca gggcgccggg ccccacttct gagcacagag cagagacaga cgcaggcggg gacaaaggca gaggatgtag ccccattggg gaggggtgga ggaaggacat gtaccctttc atgcctacac acccctcatt aaagcagagt cgtggcatct caExample 4In vitro knockdown of human ANGPTL8 in AAV-ANGPTL8 humanized mouse primary hepatocytes (MPH) with GalNAc-conjugated ANGPTL8 siRNA

[0172] Knockdown of ANGPTL8 expression by the GalNAc-conjugated ANGPTL8 siRNA was assayed using the following procedure: mouse primary hepatocytes (MPH) were freshly isolated from an AAV-ANGPTL8 humanized mouse, added to Coming 96-well plates at 15,000 cells per well, and siRNA were added directly to the well. For single point (SP) screening, 1 uM (1,000 nM) of GalNAc-conjugated siRNA was used. To generate concentration / dose response curves final concentrations of 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM of GalNAc-conjugated siRNA concentration were used.

[0173] Treated cells were lysed and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research) directly into the 96 well plate. The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and usingthe following steps in a thermocycler: 37°C for 30 minutes, 95°C for 5 minutes, and 4°C hold. Polymerase Chain Reaction (PCR) was perfoimed via TaqMan RT PCR (Life Technologies) using the following cycles temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0174] The human ANGPTL8 levels were normalized to mouse RplpO (Life Technologies) and represent the relative knockdown of human ANGPTL8 mRNA expression as compared to vehicle-treated control cells. IC50 values were calculated using a 4-parameter fit model using XLFit.

[0175] Table 6 shows the result of a single dose screen in AAV-ANGPTL8 humanized mouse primary hepatocytes by free uptake with the indicated GalN Ac-conjugated ANGPTL8 siRNA. Data were expressed as percent of message knockdown relative to untreated cells. The IC50 and percent maximum knockdown of top hits from single point screening followed by concentration / dose response curves are included as well.Table 6Percent knockdown of single dose screen and IC50 with percent maximum knockdown of top hits from single point screen in AAV-ANGPTL8 humanized mouse primary hepatocytesExample 5In vitro knockdown of human ANGPTL8 in Hep3B cells with GalNAc-conjugated ANGPTL8 siRNA

[0176] Knockdown of ANGPTL8 expression by the GalNAc-conjugated ANGPTL8 siRNA was assayed using the following procedure: transfection reagent RNAiMAX (Life Technologies) at 0.3 l / wcll was mixed with siRNA in Corning 96-well plates before adding Hep3B cells (ATCC) at 8,000 cells per well. To generate concentration / dose response curves final concentrations of 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.137, 0.046, 0.015, 0.005, and 0.0017 nM of GalNAc-conjugated siRNA concentration was used.

[0177] Treated cells are lysed and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research) directly into the 96 well plate. The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and using the following steps in a thermocycler: 37°C for 30 minutes, 95°C for 5 minutes, and 4°C hold. Polymerase Chain Reaction (PCR) was performed via TaqMan RT PCR (Life Technologies) using the following cycles temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0178] The human ANGPTL8 levels were normalized to human RPLP0 (Life Technologies) and represent the relative knockdown of human ANGPTL8 mRNA expression as compared to vehicle-treated control cells. IC50 values were calculated using a 4-paramctcr fit model using XLFit.

[0179] Table 7 shows the result of IC50 and percent maximum knockdown calculated from concentration / dose response curves in Hep3B cells by transfection with the indicatedANGPTL8 siRNA. In most instances, duplexes resulted in over 90% knockdown, with nanomolar or lower IC50.Table 7: ICso and percent maximum knockdown of ANGPTL8 message in Hep3B cells by transfection reagent, RNAiMAX, with the indicated ANGPTL8 siRNAExample 6 hi vivo Single dose Screen

[0180] GalNAc-siRNA (n=16) were tested in male C57bl / 6 mice (Taconic farms). The siRNAs were tested in a single study. Mice were dosed by retro-orbital injection with an adeno-associated virus (AAV) vector containing a plasmid with an albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7) (Vector BioLabs). Blood was collected from mice via retro-orbital sinus 14 days post AAV administration. Serum was prepared from blood and triglycerides were measured utilizing a COBAS clinical chemistry analyzer (Roche) and ANGPTL4 / 8 was measured by ELISA (Meso Scale Diagnostics). Body weight of mice were measured 22 days after AAV administration. Mice were assigned to groups with similar body weight, serum triglyceride levels, and serum ANGPTL4 / 8 (n=6 / group). Either PBS or GalNac-siRNA test article, at a dose of 5mg / kg, was administered subcutaneously to mice. Seven days post subcutaneous injection, blood was collected from all mice and serum is analyzed for triglycerides. Fourteen days post subcutaneous injection mice were euthanized under isoflurane anesthesia, blood was collected, and serum was analyzed for triglycerides. Liver was collected from the mice and frozen in liquid nitrogen. Triglyceride as a percent change from time-matched PBS was calculated as ((triglyceride minus triglyceride of PBS group ) / ( triglyceride of PBSgroup))* 100. Livers were homogenized in TriZol (Invitrogen) using Lysing Matrix D bead tubes on a FastPrep-24 (MP Bio). Chloroform was added and the aqueous phase is mixed with ethanol to precipitate the RNA. RNA was isolated on columns using PureLink Pro96 Total RNA purification kit (Invitrogen) according to manufacturer’s protocol and quantified on a NanoDrop (ThermoFisher). Equal amounts (lug) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) on Mastercycler Nexus (Eppendorf). Thermocycler settings were 25 °C for 10 min, 37 °C for 2 hrs, then 85°C for 5 min. Template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probesets and RT-PCR was performed on the QuantStudio Pro7 (ThermoFisher) with the following parameters: 50°C for 2min, 95°C for 10 minutes, then 40 cycles of 95°C for 15sec and 60°C for Imin. Fold changes (FC) were calculated as follows: the CT value of mouse RplpO was subtracted from CT value of human ANGPTL8 to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (average of PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base 2 of the negative delta delta CT value. Percent mRNA remaining was calculated multiplying the fold change by 100. Data is shown in Table 8.Table 8: C57 mice hANGPTL8 AAV in vivo single dose screenExample 7In vivo Durability 8 week

[0181] GalNAc-siRNA (n=l 1) were tested in male mice transgenic for human cholesterol ester transfer protein (CETP) and human apolipoprotein Al (Taconic farms). The siRNAs were divided and tested in 2 studies (n=7 and n=4). Mice were dosed by retro-orbital injection with two adeno-associated vims (AAV) vectors. One vector contained a plasmid with an albumin promoter and the coding sequence for human ANGPTL8(NM_018687.7) (Vector BioLabs). The second vector contained a mouse codon optimized sequence of human ANGPTL3(NP_055310.1) (Vector BioLabs). Blood was collected from mice 3-4 weeks post AAV administration. This was considered the baseline blood collection. Serum was prepared from blood and ANGPTL3 / 8 was measured by an ELISA (Meso Scale Diagnostics) and triglycerides were measured as described. Body weight of mice was measured and mice were assigned to groups with similar body weight, serum triglyceride, and ANGPTL3 / 8 levels (n=9 / group). 4-5weeks post AAV administration, either PBS or test article GalNac- siRNA, at doses 1.75 and lOmg / kg were administered subcutaneously to mice. At 2 weeks post siRNA administration, 3 mice from each group were euthanized under isoflurane anesthesia, blood was collected, and serum was analyzed for triglycerides. Liver was collected from the mice and frozen in liquid nitrogen. Blood was collected from the remaining mice (n=6 / group) at 2, 4, and 6 weeks post siRNA administration under isoflurane anesthesia. Serum was prepared from blood and triglycerides are measured. At 8 weeks post siRNA administration, the remaining mice were euthanized under isoflurane anesthesia.Blood and liver were collected from mice. Livers were processed and mRNA remaining was calculated as described in the in vivo single dose screen. Triglyceride as a percent change from PBS is calculated as described in the in vivo single dose screen.Table 9: In vivo Durability 8 week Dose Response (C57 mice hANGPTL8 AAV)Example 8In vivo Durability 15week (Dose Response (C57 mice hANGPTL8 AAV))

[0182] GalNAc-siRNA (n=5) were tested in male mice transgenic for human cholesterol ester transfer protein (CETP) and human apolipoprotein Al (Taconic farms). The siRNAs were tested in a single study. Mice were dosed by retro-orbital injection with two adeno- associated virus (AAV) vectors. One vector contained a plasmid with an albumin promoter and the coding sequence for human ANGPTL8(NM_018687.7) (Vector BioLabs). The second vector contained a mouse codon optimized sequence of human ANGPTL3(NP_055310.1) (Vector BioLabs). Blood was collected from mice 3weeks post AAV administration. This was considered the baseline blood collection. Serum was prepared from blood and triglycerides and ANGPTL3 / 8 was measured. Body weight of mice was measured and mice were assigned to groups with similar body weight, serum triglyceride and ANGPTL3 / 8 levels (n=10 / group). 4weeks post AAV administration, either PBS or test article GalNac-siRNA, at doses 0.3, 1.75 and lOmg / kg were administered subcutaneously to mice. At 2 weeks post siRNA administration, 3 mice from each group were euthanized under isoflurane anethesia, blood was collected, and serum was analyzed for triglycerides. Liver was collected from the mice and frozen in liquid nitrogen. Blood was collected from the remaining mice (n=7 / group) at 3, 6, 9, and 12wccks post siRNA administration under isoflurane anesthesia. Serum was prepared from blood and triglycerides are measured. At 15 weeks post siRNA administration, the remaining mice were euthanized under isoflurane anesthesia. Blood and liver were collected from mice. Serum was prepared from blood and triglycerides were measured. Livers were processed and mRNA remaining was calculated as described in the in vivo single dose screen. Triglyceride as a percent change from PBS was calculated as described in the in vivo single dose screen.Table 10: Dose Response (C57 mice hANGPTL8 AAV)Example 9 ANGPLT8-PrON siRNA: Liver AAV in CETPA1

[0183] Eight GalNAc-siRNAs were tested in male mice transgenic for human cholesterol ester transfer protein (CETP) and human apolipoprotein Al (Taconic farms). Mice intended for test article administration were dosed by retro-orbital injection with two adeno-associated virus (AAV) vectors. One vector contained a plasmid with an albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7) (Vector BioLabs). The second vector contained a plasmid with an albumin promoter and the mouse codon optimized sequence of human ANGPTL3 (NP_055310.1) (Vector BioLabs). Four control mice were dosed by retro-orbital injection with an AAV containing a plasmid with an albumin promoter and the firefly luciferase to establish control levels of serum triglyceride. Blood was collected from mice 4 weeks post AAV administration. This was considered the baseline blood collection. Serum was prepared from blood, and triglycerides were measured utilizing a COBAS clinical chemistry analyzer (Roche). ANGPTL3 / 8 serum protein was measured by an in-house ELISA (Meso Scale Diagnostics). Mice were assigned to nine groups with similar serum triglyceride and ANGPTL3 / 8 levels and body weight (n=9). Each group was then randomized again into two groups of n-6 (used for durability) and n=3 (used for maximum knock down at 2 weeks post dose) using the same parameters. Either PBS (Control) or test article GalNac-siRNA at a dose of 1.75mg / kg was administered subcutaneously to mice. The four control mice with the firefly luciferase AAV were untreated with PBS or test articles. At 2 weeks post siRNA administration, n=3 mice were euthanized under isoflurane anesthesia and liver (frozen in liquid nitrogen) and blood were collected to determine maximum knockdown. Blood was collected from the n=6 mice at 3, 6, 9, and 12 weeks post siRNA administration under isoflurane anesthesia. At 15 weeks post siRNA administration, remaining mice were euthanized under isoflurane anesthesia and liver (frozen in liquid nitrogen) and blood was collected. Serum was prepared from blood and triglycerides at all timepoints were measured as before. Livers were homogenized in TriZol (Invitrogen) using Lysing Matrix D bead tubes on a FastPrep-24 (MP Bio). Chloroform was added and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated on columns using RNeasy 96 Universal Tissue kit (Qiagen) according tomanufacturer’s protocol and quantitated on the NanoDrop (ThermoFisher). Equal amounts (lug) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) on Mastercycler Nexus (Eppendorf). Thermocycler settings were 25°C for 10 min, 37°C for 2 hrs, then 85°C for 5 min. Template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probesets (human Angptl8 Hs00218820_ml and mouse RPLPO MmOl 974474_gH) and RT-PCR was performed on the ABI Quant Studio 7 Pro (Applied Biosystems) with the following parameters: 50°C for 2 min, 95°C for 10 min then 40 cycles of 95°C for 15 sec and 60°C for 1 min. Fold changes (FC) were calculated as follows: the CT value of mouse RplpO was subtracted from CT value of human ANGPTL8 to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base 2 of the negative delta delta CT value. Percent knock down (%KD) was calculated by subtracting FC from one and multiplying by 100 and shown in Table 12. Triglyceride and mRNA fold change data was analyzed for a statistically significant difference from the PBS group at corresponding timepoint using ANOVA and Dunnett’s method where p < 0.05 was considered statistically significant (SAS Institute) Data is shown in Table 11.Table 11: Triglyceride reduction at indicated week following a 1.75 mg / kg subcutaneous dose expressed as percent change vs PBS (numbers in bold indicate p<0.05 by ANOVA with Dunnett’s)Table 12: hAngplt8 AAV gene knockdown data measured at 2- and 15-weeks post dose (RPLPO as housekeeping gene)

Claims

CLAIMSWhat is claimed is:

1. An ANGPTL8 RNAi agent, or a salt thereof, wherein the RNAi agent comprises an antisense strand and a sense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises the formula:wherein:R1is selected from the group consisting of H, OR2, NH2, SR2, F, and Cl, wherein R2is selected from the group consisting of H and C1-C20 alkyl; andB is a nucleobase; wherein the antisense strand comprises at least 15 nucleotides of any one of SEQ ID NOs 6, 7, 8, 9, 10, or a sequence having 90% sequence identity thereto, or 15 nucleotides of an antisense strand sequence as set forth in any one of Tables 2A, 2B, 3A, 3B, 4, and 5, or a sequence having 90% sequence identity thereto.

2. The ANGPTL8 RNAi agent, or a salt thereof, of claim 1, comprising the formula:wherein: each Xi is an independently selected nucleotide;each X2 is an independently selected nucleotide;Q is PO2X5, wherein X5is O or S, or, when n is 0, Q may be H; and m and n are each independently selected from any integer from 0 to 24, such that the sum of m+n is between 14 and 24 inclusive; wherein each B is independently selected.

3. The ANGPTL8 RNAi agent, or salt thereof, of claim 1 or claim 2, wherein the sense strand is 21 to 25 nucleotides in length.

4. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 3, wherein the antisense strand is between 21 and 23 nucleotides in length.

5. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 4, wherein the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

6. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 5, wherein the duplex region comprises a region of complementarity which is at least 18 nucleotides in length.

7. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 6, wherein the duplex region between the sense strand and the antisense strand comprises 0, 1, or 2 mismatches between the sense strand and the antisense strand.

8. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 7, wherein the duplex region between the sense strand and the antisense strand comprises 0 mismatches between the sense strand and the antisense strand.

9. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 8, wherein the antisense strand comprises 15 contiguous nucleotides of any one of SEQ ID NOs: 6, 7, 8, 9,10, 11, 12, 13, 14, or 15, or a sequence of Tables 3A, 3B, 4 and 5.

10. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 9, wherein the antisense strand comprises 18 contiguous nucleotides of any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a sequence of Tables 3A, 3B, 4 and 5.

11. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 10, wherein the sense strand is selected from the group consisting of SEQ ID NOs: 1 to 5, or a sequence having at least 90% sequence identity thereto, or a sense strand sequence set forth in one of Tables 3 A, 3B, 4, and 5, or a sequence having 90% sequence identity thereto.

12. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 11, wherein the antisense strand comprises the formula:

13. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 12, wherein the antisense strand comprises the formula:

14. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 13, wherein the antisense strand comprises the formula:

15. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 12, wherein the antisense strand comprises the formula:wherein X5is selected from O and S.

16. The ANGPTL8 RNAi agent, or salt thereof, of claim 15, wherein the antisense strand comprises the formula:

17. The ANGPTL8 RNAi agent, or salt thereof, of claim 15, wherein the antisense strand comprises the formula:

18. The ANGPTL8 RNAi agent, or salt thereof, of claim 15, wherein the antisense strand comprises the formula:

19. The ANGPTL8 RNAi agent, or salt thereof, of claim 15, wherein the antisense strand comprises the formula:

20. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 12, wherein the antisense strand comprises the formula:

21. The ANGPTL8 RNAi agent, or salt thereof, of claim 20, wherein the antisense strand comprises the formula:

22. The ANGPTL8 RNAi agent, or salt thereof, of claim 20 or claim 21, wherein the antisense strand comprises the formula:

23. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 20 to 22, wherein the antisense strand comprises the formula:

24. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 20 to 22, wherein the antisense strand comprises the formula:

25. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 20 to 22, wherein the antisense strand comprises the formula:

26. The ANGPTL8 RNAi agent, or salt thereof, of claim 25, wherein the antisense strand comprises the formula:

27. The ANGPTL8 RNAi agent, or salt thereof, of claim 25, wherein the antisense strand comprises the formula:

28. The ANGPTL8 RNAi agent, or salt thereof, of claim 25, wherein the antisense strand comprises the formula:

29. The ANGPTL8 RNAi agent, or salt thereof, of claim 25, wherein the antisense strand comprises the formula:

30. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 29, wherein m and n are each independently selected from any whole number from 0 to 20.

31. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 30, wherein the oligonucleotide has one of a phosphate, a hydroxyl, a phosphate analog, an abasic moiety, and an inverted abasic moiety at its 5’ end.

32. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 31, wherein each B is independently selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine,5-hydoxymethylcytosine, dihydrouridine, 5 -methylcytidine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

33. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 32, wherein each B is independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

34. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 33, wherein each B is uracil.

35. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 34, wherein the antisense strand is 23 to 30 nucleotides in total length.

36. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 35, wherein the antisense strand is 23 nucleotides in total length.

37. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 36, comprising at least one modification selected from the group consisting of 2’-O-mcthoxy, 2’-O-mcthyl, 2’ -fluoro, phosphorothioate, 2’ -deoxy, abasic moiety, inverted abasic moiety, R-PrON, and S- PrON.

38. The ANGPTL8 RNAi agent, or salt thereof, of claim 37, wherein the antisense strand comprises 2’ -fluoro modified nucleotide at the following positions: a. ff39. The ANGPTL8 RNAi agent, or salt thereof, of claim 38, wherein the antisense strand is an 2’0-methyl modified nucleotide at all other positions.

40. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 39, wherein the sense strand and antisense strand each independently comprise one or more modifiedintemucleotide linkages, and wherein each modified intemucleotide linkage is a phosphorothioate linkage.

41. The ANGPTL8 RNAi agent, or salt thereof, of claim 1 to 40, wherein the sense strand and antisense strand each independently comprise four phosphorothioate linkages.

42. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 41, wherein the 5’ terminal nucleotide of the antisense strand comprises an OH group, a phosphate group, a vinyl phosphonate, or a phosphate analog.

43. The ANGPTL8 RNAi agent, or salt thereof, of claim 42, wherein the 5’ terminal nucleotide of the antisense strand is further modified to replace the 5’ phosphate group with an OH group.

44. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1-43, wherein the antisense strand has a 3’ overhang of 1 or 2 nucleotides.

45. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1-44, further comprising a delivery moiety conjugated to the RNAi agent.

46. The ANGPTL8 RNAi agent, or salt thereof, of claim 45, wherein the delivery moiety is at the 3’ end of the sense strand.

47. The ANGPTL8 RNAi agent, or salt thereof, of claim 45 or claim 46, wherein the delivery moiety is a GalNAc delivery moiety.

48. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 45 to 47, wherein the delivery moiety comprises the formula:wherein E is a point at which the delivery moiety is conjugated to the sense strand.

49. The ANGPTL8 RNAi agent, or salt thereof, of claim 48, wherein the delivery moiety is conjugated directly to the sense strand.

50. The ANGPTL8 RNAi agent, or salt thereof, of claim 48, wherein the delivery moiety is conjugated to the sense strand via a linker.

51. The ANGPTL8 RNAi agent, or salt thereof, of claim 49 or claim 50, wherein the linker comprises the formula:wherein the delivery moiety is conjugated to the linker at connection point A, and the duplex RNA is conjugated to the linker at connection point B.

52. The ANGPTL8 RNAi agent, or salt thereof, of claim 49 or claim 50, wherein the linker comprises the formula:wherein the delivery moiety is conjugated to the linker at connection point X, and the duplex RNA is conjugated to the linker at connection point Y.

53. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 45 to 47, wherein the delivery moiety comprises the formula of:wherein E is a point at which the delivery moiety is conjugated to the sense strand.

54. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 53, wherein the RNAi agent decreases expression of the ANGPTL8 gene in a liver cell, as compared to a control agent.

55. The ANGPTL8 RNAi agent, or salt thereof, of claim 1 , wherein the wherein the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 516, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 519; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 516, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 520;the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 516, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 521; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 516, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 522; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 516, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 523; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 517, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 524; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 518, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 525; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 518, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 526; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 518, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 527; the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 518, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 528; and the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 518, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 529.

56. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 55, for use in therapy.

57. The ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 55, for use in the treatment of a disease or disorder selected from: cardiovascular disease, cardiometabolic disease, diabetes, dyslipidemia, aberrant renal function, hypertension, nonalcoholic fatty liver disease such as nonalcoholic steatohepatitis (NASH), and obesity.

58. The ANGPTL8 RNAi agent, or salt thereof, for use according to claim 55, wherein the disease or disorder is dyslipidemia.

59. A pharmaceutical composition comprising the ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 55, and one or more pharmaceutically acceptable excipients.

60. Use of the ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 55, in the manufacture of a medicament for the treatment of a disease or disorder selected from: cardiovascular disease, diabetes, dyslipidemia, aberrant renal function, hypertension, nonalcoholic fatty liver disease such as nonalcoholic steatohepatitis (NASH), and obesity.

61. The use of claim 60, wherein the disease or disorder is dyslipidemia.

62. A method of treating dyslipidemia in a patient in need thereof, comprising administering the ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 55, or a pharmaceutical composition thereof, to the patient.

63. A method of decreasing ANGPTL8 expression in a cell, comprising contacting the cell with the ANGPTL8 RNAi agent, or salt thereof, of any one of claims 1 to 55.

64. The method of claim 63, wherein the method further comprises incubating the cell for a time sufficient for decreasing the level of ANGPTL8 mRNA by at least 50% as compared to an untreated cell.

Images