Novel RNA therapeutics and uses thereof

Abstract

The present invention relates to novel therapeutic compounds, referred to as RNAi agents, which reduce the expression of the ANGPTL8 receptor (expressed by the ANGPTL8 gene), thereby reducing mRNA expression and protein expression. Such RNAi agents are useful in the treatment of diseases involving modulation of ANGPTL8 expression and function, such as dyslipidemia, cardiovascular diseases or cardiac metabolic diseases.

Description

background

[0001] The present disclosure relates to novel RNAi agents designed to reduce ANGPTL8 expression in the liver, wherein the RNAi agents comprise a delivery moiety optionally conjugated to an oligonucleotide via a linker. These RNAi agents can be used to treat diseases involving modulation of ANGPTL8 expression.

[0002] Angiopoietin-like protein 8 (ANGPTL8) is primarily expressed in the liver and adipose tissue and plays an important role in triglyceride metabolism. ANGPTL8, along with ANGPTL3 or ANGPTL4, is thought to regulate triglyceride levels by inhibiting the enzymatic activity of lipoprotein lipase (LPL). When LPL is active, it hydrolyzes triglycerides and reduces circulating plasma triglyceride levels. Elevated ANGPTL8 levels have been observed or associated with cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), renal dysfunction, hypertension, nonalcoholic fatty liver disease such as nonalcoholic steatohepatitis (NASH), and obesity.

[0003] ANGPTL8 siRNA and ASO have been described, such as those disclosed in WO2020 / 104649A2, but have not progressed in patient treatment. Herein, the use of ANGPTL8 RNAi agents to reduce ANGPTL8 expression can be used to treat cardiovascular metabolic and related diseases, such as dyslipidemia, in patients in need. SUMMARY OF THE INVENTION

[0004] In one aspect, provided herein are RNAi agents for reducing expression of an ANGPTL8 gene, wherein the RNAi agent comprises a delivery moiety of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand:

[0005]

[0006] wherein R is conjugated to the attachment point E of Formula I, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises a region complementary to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified internucleotide linkages. In some embodiments, Formula I is optionally conjugated to the sense strand via a linker. In some embodiments, Formula I is optionally conjugated to the 3' terminal nucleotide of the sense strand via a linker.

[0007] In one embodiment, provided herein is an RNAi agent for reducing expression of an ANGPTL8 gene, wherein the RNAi agent comprises a delivery moiety of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand:

[0008]

[0009] Wherein R is conjugated to the connection point E of formula I, optionally conjugated via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or a sequence with 90% sequence identity thereto, or an antisense strand sequence shown in Tables 3A, 3B and 4, or a sequence with 90% sequence identity thereto, and wherein the sense strand and antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified internucleotide linkages. In some embodiments, Formula I is optionally conjugated to the sense strand via a linker. In some embodiments, Formula I is optionally conjugated to the 3' terminal nucleotide of the sense strand via a linker.

[0010] 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 18 to 23 nucleotides in length. In some embodiments, the sense strand is 18 to 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

[0011] In some embodiments, the sense strand or the antisense strand comprises a sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 disclosed herein. In some embodiments, both the sense strand and the antisense strand comprise a sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 disclosed herein.

[0012] In some embodiments, R is conjugated to Formula I via a linker. In further embodiments, the linker comprises a linker of Formula II having attachment points A and B, or a linker of Formula III having attachment points C and D, and wherein:

[0013]

[0014] a. Formula I is conjugated to attachment point A of Formula II at attachment point E, and Formula II is conjugated to a phosphate group at attachment point B, and the phosphate group is further conjugated to R; or

[0015] b. Formula I is conjugated to attachment point C of Formula III at attachment point E, and Formula III is conjugated to a phosphate group at attachment point D, and the phosphate group is further conjugated to R.

[0016] In another aspect, provided herein is a pharmaceutical composition comprising an 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), abnormal renal function, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), or obesity in a patient in need thereof, comprising administering to the patient an ANGPTL8 RNAi agent described herein, or a pharmaceutical composition thereof.

[0018] In another aspect, provided herein are ANGPTL8 RNAi agents for use in therapy. Also provided herein are uses of ANGPTL8 RNAi agents in the preparation of medicaments for treating cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), abnormal renal function, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), or obesity. DETAILED DESCRIPTION

[0019] Such siRNAs may exhibit one or more of the following properties, for example, compared to other liver-targeted siRNAs (e.g., ANGPTL8 siRNAs) comprising different delivery ligands, different sequences, or different modified sequences, or compared to vehicle control treatment: improved knockdown in the liver; improved tissue exposure; improved hepatocyte exposure; improved durability of response; improved pharmacokinetic properties; reduced off-target effects; and / or improved toxicity profiles. Other embodiments of ANGPTL8 RNAi drugs herein may include one or more of the following properties: fewer side effects compared to statins or other standard therapies; improved toxicity profiles; improved safety profiles; improved tolerability or compliance; and / or improved liver function tests. Other siRNAs herein may have other benefits, for example, in combination with any of the above benefits or as independent benefits, including improved and / or simplified synthesis, synthesis processes with fewer degradation products; or any combination thereof.

[0020] The RNAi reagent herein comprises a sense strand and an antisense strand, wherein each strand is an oligonucleotide. In some embodiments, the RNAi reagent herein further comprises a delivery moiety. As used herein, "nucleotide" refers to an organic compound having a nucleoside (nucleobase, such as adenine, cytosine, guanine, thymine or uracil; and a pentose, such as ribose or 2-deoxyribose) and a phosphate group. "Nucleotide" can be used as a monomeric unit of a nucleic acid polymer, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

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

[0022] As used herein, "ribonucleotide" refers to a nucleotide containing ribose as its pentose sugar and containing a hydroxyl group at the 2' position. A modified ribonucleotide refers to a ribonucleotide having one or more modifications or substitutions at the 2' position with atoms other than hydrogen, including modifications or substitutions internal or external to the nucleobase, sugar group, or phosphate group.

[0023] As used herein, "modified internucleotide linkage" refers to an internucleotide linkage having one or more chemical modifications compared to a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage may be a non-naturally occurring linkage.

[0024] As used herein, "modified nucleotides" refer to nucleotides having one or more chemical modifications compared to corresponding reference nucleotides selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides and thymine deoxyribonucleotides. Modified nucleotides can be non-naturally occurring nucleotides. For example, modified nucleotides can have one or more chemical modifications in their sugar groups, core bases and / or phosphate groups. In addition, or as another option, modified nucleotides can have one or more chemical moieties conjugated to corresponding reference nucleotides.

[0025] The term "percentage of sequence identity" is defined for a reference nucleic acid sequence as follows: after the best comparison candidate sequence and the reference nucleic acid sequence (a gap (gap) or overhang (overhang) can be introduced to maximize the percentage of sequence identity), the percentage of nucleotides, nucleosides or bases identical with the reference nucleic acid sequence in the candidate sequence. The sequence alignment for determining the percentage of nucleotide sequence identity can be achieved in a variety of ways well known to those skilled in the art, such as using disclosed computer software programs, such as Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), including BLAST, BLAST-2, ALIGN, Clustal W 2.0 or Clustal X 2.0 or Megalign (DNASTAR) software. In one embodiment of the invention, sequence identity is calculated using Clustal W 2.0 or Clustal X 2.0. In another embodiment, sequence identity is calculated using Clustal W 2.0. In another embodiment, Clustal X2.0 is used to calculate sequence identity. Those skilled in the art can determine the appropriate parameters for measuring comparison, including any algorithm required for maximum comparison on the full length of the sequence being compared. The percentage of " sequence identity " can be determined by comparing the sequences of two optimal comparisons in a comparison window, wherein, compared with a reference sequence (not comprising an addition or deletion), the nucleic acid sequence fragment in the comparison window may comprise an addition or deletion (for example, a gap or an overhang), to achieve the optimal comparison of two sequences. The number of sites where identical nucleotides, nucleosides or core bases occur in the two sequences can be determined to obtain the matching site number, the matching site number is divided by the total number of sites in the comparison window, and the result is multiplied by 100 to obtain the percentage of sequence identity, thereby calculating the percentage. The output is the percentage identity of the test sequence relative to the query sequence. In certain embodiments, the percentage identity of the sequence refers to the percentage of the identical nucleotide residues between the two chains calculated using PID3, i.e., the identical nucleotide residue number is divided by the total number of nucleotides of the shortest sequence in the two sequences, then multiplied by 100. See, eg, Raghava, G., Barton, GJ Quantification of the variation in percentage identity for protein sequence alignments. BMCBioinformatics 7, 415 (2006).

[0026] As used herein, "phosphate analogue" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analogue is located at the 5' terminal nucleotide of the oligonucleotide, replacing the 5' phosphate. The 5' phosphate analogue may comprise a phosphatase-resistant connection. Examples of phosphate analogues include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). The oligonucleotide may have a phosphate analogue (referred to as a "4' phosphate analogue") at the 4' carbon position of the sugar of the 5' terminal nucleotide. An example of a 4' phosphate analogue is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to a sugar moiety (e.g., at its 4' carbon) or an analog thereof. See, for example, International Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed to the 5' end of oligonucleotides (see, eg, International Patent Application Publication 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" refers to a nucleotide sequence of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence so that hybridization occurs between the two nucleotide sequences under appropriate hybridization conditions (e.g., in phosphate buffer, in cells, etc.). In some embodiments, the oligonucleotides herein include a targeting sequence having a region complementary to an mRNA target sequence.

[0028] As used herein, the term "duplex" refers to a structure formed by hydrogen bonds of complementary base pairing of two antiparallel nucleotide sequences under suitable conditions when referring to nucleic acids or oligonucleotides (such as sense or antisense strands) to promote the formation of such structures. Although the two chains may not be completely complementary, or there are no basic nucleotides, duplexes can still form. As shown herein, for example, in Table 2A, Table 2B, Table 3A, Table 3B or Table 4, duplex No: corresponds to a specific sense and antisense strand comprising a given RNAi agent.

[0029] RNA interference is a specific cellular process that utilizes RISC to degrade RNA in a sequence-dependent manner. As used herein, an "RNAi agent" comprises (a) a double-stranded oligonucleotide having a sense strand (passenger strand) and an antisense strand (guide strand), wherein the antisense strand or a portion of the antisense strand is used by an Argonaute 2 (Ago2) endonuclease to cleave a target mRNA, or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand (or a portion of the antisense strand) is used by an Ago2 endonuclease to cleave a target mRNA. In some embodiments, the RNAi agent comprises a delivery portion.

[0030] As used herein, The bond indicated represents the point of attachment as described herein. For example, if a general variable (e.g., X) is stated to be attached at the attachment point E shown below, it is intended that X be bonded to the atom at the attachment point (see the scheme below).

[0031]

[0032] As used herein, "treatment" or "treating" refers to any process that may slow, control, delay, or prevent the progression of a disease or condition disclosed herein, or improve the symptoms of a disease or condition, and does not necessarily mean that all symptoms of the disease or condition are completely eliminated. Treatment includes administering an RNAi agent or a pharmaceutical composition thereof to treat a disease or condition in a mammal (including a human).

[0033] An "effective amount" refers to the amount required (over a period of time and manner of administration) to achieve the desired therapeutic effect. The effective amount of an RNAi agent may vary depending on factors such as the individual's disease state, age, sex, weight, and the ability of the RNAi agent to elicit the desired response in the individual. An effective amount also refers to an amount in which any toxic or deleterious effects of the RNAi agent are offset by its therapeutic benefit.

[0034] Provided herein are RNAi agents for reducing expression of an ANGPTL8 gene, wherein the RNAi agent comprises a delivery moiety of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand:

[0035]

[0036] wherein R is conjugated at attachment point E of Formula I, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises a region complementary to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense strand and antisense strand each optionally comprise one or more modified nucleotides and linkages between the one or more modified nucleotides.

[0037] Also provided herein are RNAi agents for reducing expression of an ANGPTL8 gene, wherein the RNAi agent comprises a delivery moiety of Formula Ia conjugated to R, wherein R comprises an antisense strand and a sense strand:

[0038]

[0039] wherein R is optionally conjugated to Formula Ia via a linker, wherein the sense and antisense strands form a duplex region, and wherein the antisense strand comprises a region complementary to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense and antisense strands each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages. Disclosed herein are RNAi agents for reducing ANGPTL8 gene expression, wherein the RNAi agent comprises a sense and antisense strand, wherein the sense and antisense strands form a duplex region, and wherein the antisense strand comprises at least 15 nucleotides as described in the antisense strand sequences disclosed herein, and wherein the sense and / or antisense strands 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 3A, Table 3B, or Table 4. In further embodiments, the RNAi agent reduces ANGPTL8 gene expression by about 50% or more in cells expressing ANGPTL8, compared to a control. In further embodiments, the RNAi agent reduces ANGPTL8 gene expression by reducing ANGPTL8 mRNA transcript levels, ANGPTL8 protein levels, or both.

[0040] In a further embodiment, the antisense strand is 15 to 25 nucleotides in length, and / or the sense strand is 15 to 25 nucleotides in length. In a further embodiment, the antisense strand is 18 to 23 nucleotides in length. In a further embodiment, the sense strand is 18 to 21 nucleotides in length.

[0041] In further embodiments, the RNAi agent comprises at least 18 consecutive nucleotides of an antisense strand sequence described in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4.

[0042] In a further embodiment, the antisense strand of the RNAi agent is 23 nucleotides in length. In an even further embodiment, the sense strand is 21 nucleotides in length. In another embodiment, the sense strand and the antisense strand comprise a sequence selected from the group consisting of sequences shown in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4.

[0043] The sense and antisense strands in the RNAi agents disclosed herein need not be completely complementary. Thus, in the RNAi agents disclosed herein, the duplex region between the sense and antisense strands comprises 0, 1, 2, or 3 mismatches. In further embodiments, the duplex region between the sense and antisense strands consists of 0, 1, 2, or 3 mismatches between the sense and antisense strands.

[0044] 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 yet further embodiments of the RNAi agents disclosed herein, each nucleotide of the sense strand and each nucleotide of the antisense strand are modified nucleotides. In further embodiments, each nucleotide is a 2' fluoro-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0045] In further embodiments, the antisense strand has an antisense strand sequence shown in Table 2A, Table 2B, or Table 4, or a sequence having at least 90% sequence identity thereto, or an antisense strand sequence shown in Table 3A or Table 3B, or a sequence having at least 90% sequence identity thereto. In other embodiments, the antisense strand sequence or sense strand sequence shown in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4, independently, is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0046] 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 nucleotide may occur at a position different from that shown in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4. In one embodiment, the 2' fluoro-modified nucleotide is present at

[0047] a. positions 2, 3, 7, 14 and 16 from the 5' end of the antisense strand; or

[0048] b. positions 2, 5, 7, 14 and 16 from the 5' end of the antisense strand; or

[0049] c. positions 2, 3, 8, 14, and 16 from the 5' end of the antisense strand; or

[0050] d. positions 2, 5, 8, 14 and 16 from the 5' end of the antisense strand; or

[0051] e. Positions 2, 6, 14 and 16 from the 5' end of the antisense strand.

[0052] In a further embodiment, the nucleotides other than 2' fluoro-modified nucleotides are 2'-O-methyl-modified nucleotides.

[0053] In a further embodiment of the RNAi reagent disclosed herein, the sense strand and the antisense strand each independently comprise a connection between one or more modified nucleotides, and the connection between each modified nucleotide is a phosphorothioate connection. In a further embodiment, the sense strand and the antisense strand each independently comprise four phosphorothioate connections. In a further embodiment, the two terminal nucleotides of the 5' end and the 3' end of the sense strand and the antisense strand are phosphorothioate connections.

[0054] 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, a "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is located at the 5' terminal nucleotide of an oligonucleotide, replacing a 5' phosphate. The 5' phosphate analog may comprise a phosphatase-resistant connection. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). An oligonucleotide may have a phosphate analog (referred to as a "4' phosphate analog") at the 4' carbon position of the sugar of the 5' terminal nucleotide. An example of a 4' phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bound to a sugar moiety (e.g., at its 4' carbon) or an analog thereof. See, for example, International Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed to the 5' end of oligonucleotides (see, eg, International Patent Application Publication No. WO 2011 / 133871; US Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0055] In further embodiments, the 5'-terminal nucleotide of the antisense strand may comprise a further modification, wherein the 5'-terminal nucleotide contains a vinylphosphonate, a phosphate, or a hydroxyl group as the 5'-terminal group. In other embodiments, the phosphate at the 5'-terminus of SEQ ID NO: is removed and replaced with an OH group. In other embodiments, the phosphate at the 5'-terminus of SEQ ID NO: is replaced with a 5' vinylphosphonate group.

[0056] In further embodiments, 1, 2 or 3 mismatches are introduced into the sense strand sequence in Table 2A, Table 2B, Table 3A, Table 3B. In further embodiments, 1, 2 or both terminal nucleotides at the 5' end of the antisense strand are altered.

[0057] In some embodiments of the RNAi agents described herein, the antisense strand comprises a first nucleic acid sequence that has at least 90% sequence identity to the antisense sequence corresponding to duplex No. 1 in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4, and the sense strand comprises a second nucleic acid sequence that has at least 90% sequence identity to the sense sequence corresponding to the same duplex No. 1 in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4. For example, in one embodiment, the antisense strand comprises a first nucleic acid sequence that has at least 90% sequence identity to the antisense sequence corresponding to duplex No. 1 in Table 2A, i.e., 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 the sense sequence corresponding to duplex No. 1 in Table 2A (i.e., SEQ ID NO: 1). In further embodiments, the 5' phosphate group of the antisense strand is further modified / replaced with a 5' vinylphosphonate or OH group.

[0058] In a further embodiment, 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.

[0059] In other embodiments disclosed herein, the RNAi agent has a delivery moiety of Formula I conjugated to R:

[0060]

[0061] wherein R is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that are complementary to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511 and the sense strand and 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 antisense strand each independently comprise one or more modified internucleotide linkages, and wherein R is optionally conjugated to Formula I via a linker. In further embodiments, the sense strand or the antisense strand is selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 disclosed herein. In other embodiments, the antisense or antisense strand of the RNAi agent has an antisense strand sequence and / or sense strand sequence that has 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 a corresponding sequence selected from Table 2A, Table 2B, Table 3A, Table 3B or Table 4 herein.

[0062] In other embodiments, the RNAi agents disclosed herein comprise a linker. In other further embodiments, R is conjugated to Formula I via a linker. In yet further embodiments, the linker comprises a linker of Formula II having connection points A and B, or a linker of Formula III having connection points C and D, and wherein:

[0063]

[0064] a. The RNAi agent comprises Formula I, Formula I is conjugated to Linker Point A of Formula II at Linker Point E, and Formula II is conjugated to a phosphate group at Linker Point B, and the phosphate group is conjugated to R; or

[0065] b. The RNAi agent comprises Formula I, Formula I is conjugated to the attachment point C of Formula III at the attachment point E, and Formula III is conjugated to a phosphate group at the attachment point D, and the phosphate group is further conjugated to R.

[0066] In other embodiments, when the RNAi agent comprises a linker, R is conjugated to Formula I via a linker, and the linker is a linker comprising Formula III having attachment points C and D:

[0067]

[0068] And wherein the RNAi agent comprises Formula I conjugated to Formula III at connection point C, and Formula III is conjugated to a phosphate group at connection point D, and the phosphate group is further conjugated to R.

[0069] The sense and antisense strands of the RNAi agent can be synthesized using any nucleic acid polymerization method known in the art, for example, solid phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, STR et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercial synthesizers, such as the MerMade from LGC Biosearch Technologies, can be used. TM 12, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate bonds can be introduced using sulfurizing agents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylene)amino)-3H-1,2,4-dithiazoline-3-thione)). Modified oligonucleotides can be synthesized using similar techniques and commercially available modified phosphoramidites and controlled pore glass (CPG) products, as is well known.

[0070] In other embodiments, the RNAi agent is capable of reducing the expression of the ANGPTL8 gene in hepatocytes. In other embodiments, the RNAi agents disclosed herein are used for treatment. In further embodiments, the use is to treat dyslipidemia, such as elevated plasma triglyceride levels. In other embodiments, the RNAi agents disclosed herein are used to treat cardiovascular disease. In other embodiments herein, the RNAi agent is used to prevent cardiovascular events. In further embodiments, a cardiovascular event is myocardial infarction. In other embodiments, the use is to reduce the number of hospitalizations associated with cardiovascular disease or events. In other embodiments, the use is to treat non-alcoholic fatty liver disease (NAFLD). In further embodiments, NAFLD is non-alcoholic steatohepatitis (NASH). In other embodiments, the use is to reduce the inhibition of lipoprotein lipase (LPL). In further embodiments, the use is to increase the catabolism of triglyceride-rich lipoproteins. In other embodiments, the RNAi agent is used to treat liver disease in patients who may benefit from reducing the expression level of ANGPTL8. In other embodiments, the use is to treat any of the above-mentioned diseases after statin use fails to control one or more symptoms, such as failure to reduce one or more levels of total-C, LDL-C, apo B, and / or failure to increase HDL-C levels. In other embodiments, the use is to treat any of the above-mentioned diseases in patients who are intolerant to statins; in further embodiments, the use is to reduce LDL-C levels in patients who are intolerant to statins. In further embodiments, the use is for any of the above-mentioned uses after dietary changes fail to control one or more symptoms. In other further embodiments, the use is as an adjunct therapy to diet for any of the above-mentioned uses.

[0071] RNAi preparations can be formulated into pharmaceutical compositions. Accordingly, disclosed herein are pharmaceutical compositions comprising the RNAi preparations 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).

[0072] In other embodiments, the RNAi agents described herein are used in the preparation of a medicament for treating dyslipidemia, or for any of the uses described in the preceding paragraphs.

[0073] In other embodiments, methods of treating dyslipidemia in a patient in need thereof are provided, comprising administering to the patient an RNAi agent disclosed herein or a pharmaceutical composition thereof. In other embodiments, methods of treating dyslipidemia in a patient in need thereof, or any of the uses described above, or a pharmaceutical composition thereof. In other embodiments, methods of treating a patient who is unable to achieve blood lipid levels after statin and / or dietary therapy are provided, comprising administering to the patient an RNAi agent disclosed herein or a pharmaceutical composition thereof.

[0074] RNAi drugs can be administered to patients either intravenously or subcutaneously.

[0075] RNAi dosage regimens can be adjusted to optimize the desired effect (e.g., therapeutic response), for example, as a single bolus, in divided doses over time, or by proportionally increasing or decreasing the dose according to the exigencies of the therapeutic situation.

[0076] Dosage values may vary depending on the type and severity of the condition to be alleviated. In addition, 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 composition.

[0077] In other embodiments, methods are provided for reducing ANGPTL8 expression in a cell, comprising contacting the cell with an RNAi agent disclosed herein and incubating the cell for a period of time sufficient to reduce the level of ANGPTL8 mRNA by at least 50% compared to untreated or control-treated cells.

[0078] Certain abbreviations are defined as follows: “1,2-DCE” refers to 1,2-dichloroethane; “DCM” refers to dichloromethane; “DIEA” refers to N,N-diisopropylethylamine; “DMF” refers to N,N-dimethylformamide; “DMAP” refers to 4-dimethylaminopyridine; “DMTCl” refers to 4,4′-dimethoxytrityl chloride; “DPP4” refers to dipeptidyl peptidase; “EDC” refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; “EtOAc” refers to ethyl acetate; “GalNAc” refers to N-acetylgalactosamine; “HATU” refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; “HBTU” refers to O-(benzotriazol-1-yl)-N,N “Hydroxybenzotriazole” refers to 1,4-dihydro-1,4-dihydro-1,4-dihydro-2 ...2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro-2,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro-2,4-dihydro-2

[0079] Delivery moieties comprising Formula I can be prepared by the following non-limiting synthetic steps and schemes.

[0080] Solution 1

[0081]

[0082] Scheme 1, Step A depicts the cyclization of compound (1) using trimethyl triflate in a solvent such as 1,2-DCE to provide compound (2). Step B shows the addition of hex-5-en-1-ol to compound (2) using trimethylsilyl triflate in a solvent such as 1,2-DCE to provide compound (3). Step C shows the oxidation of compound (3) using a suitable oxidant such as sodium periodate and a catalyst such as ruthenium(III) chloride to provide compound (4).

[0083] Option 2

[0084]

[0085] Scheme 2, Step A shows the amide coupling of compound (5) with tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate using HBTU and HOBt with an appropriate base such as DIEA in a solvent such as DMF to provide compound (6). Step B depicts the alkaline hydrolysis of compound (6) using a base such as aqueous NaOH in a THF and MeOH solvent system to provide compound (7). Step C shows the amide coupling of compound (7) with allyl 11-aminoundecanoate hydrochloride using HATU with an appropriate base such as DIEA in a solvent such as DMF to provide compound (8). Step D shows the acidic deprotection of compound (8) using TFA in a solvent such as DCM to provide compound (9). Step E shows the amide coupling of compound (9) with compound (4) using EDC and HOBt in a solvent such as DCM to provide compound (10). Step F shows the deprotection of compound (10) using tetrakis(triphenylphosphine)palladium and PhSiH3 in a solvent such as DCM to give compound (11). Step F depicts the coupling of compound (11) with NHS using EDC in a solvent such as DCM to give compound (12).

[0086] Option 3

[0087]

[0088] Scheme 3, Steps AC are essentially similar to Steps CE in Scheme 2, starting from compound (7), to provide compounds (13), (14) and (15). Step D depicts the hydrogenation of compound (15) using a palladium / carbon catalyst in a solvent such as MeOH to provide compound (16). Step E is essentially similar to Step G in Scheme 2 to provide compound (17).

[0089] Option 4

[0090]

[0091] Scheme 4, Step AI, consists of a series of amide coupling and deprotection reactions, and the methods used are basically similar to those in Schemes 2 and 3, starting from compound (18) to obtain compound (27).

[0092] Option 5

[0093]

[0094] The method described in Scheme 5, Steps AC is basically similar to the method of Steps GI in Scheme 4, starting from compound (24) to obtain compound (30).

[0095] Option 6

[0096]

[0097] Scheme 6, Step A depicts the protection of compound (31) using DMTCl with a suitable base such as DIEA in a solvent such as DCM to provide compound (32). Step B shows the amide coupling of compound (32) and piperidin-4-ylmethanol using HBTU and HOBt with TMP in a solvent such as DCM to provide compound (33). Step C shows the deprotection of compound (33) using 20% piperidine in DMF to provide compound (34).

[0098] Option 7

[0099]

[0100] Scheme 7, Step A in Scheme 7 is essentially similar to Step A in Scheme 2, and compound (35) is obtained by coupling compounds (16) and (34). Step B shows the addition of succinic anhydride to compound (35) in a suitable solvent such as DCM in the presence of a base system of TEA and DMAP to form compound (36). Step C depicts the loading of compound (36) onto a resin using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and a base such as DIEA in a solvent system such as MeCN and DCM to provide compound (37).

[0101] Preparation 1

[0102] Methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate

[0103]

[0104] To a solution of methyl (5-acetamido-3,4,6-triacetoxy-tetrahydropyran-2-yl)acetate (9.00 g, 23.1 mmol) in 1,2-DCE (46 mL) was added trimethylsilyl trifluoromethanesulfonate (6.5 mL, 35 mmol). The mixture was heated to 50° C. and stirred for 18 hours. After this time, the mixture was diluted with DCM (200 mL), washed with saturated NaHCO 3 (200 ml) and saturated aqueous sodium chloride solution (200 mL”), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 0-10% MeOH / DCM to give the title compound (6.434 g, 84%). ES / MS m / z 330 (m+H).

[0105] Preparation 2

[0106] Methyl (5-acetylamino-3,4-diacetoxy-6-hexyl-5-phenoxytetrahydropyran-2-yl) acetate

[0107]

[0108] To a solution of methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate (30.43 g, 92.42 mmol) in 1,2-DCE (231 mL) was added hex-5-en-1-ol (22.2 mL, 185 mmol), followed by the addition of activated powdered Molecular sieves (15.6 g). The suspension was stirred at ambient temperature for 30 minutes, then trimethylsilyl trifluoromethanesulfonate (19 mL, 101.9 mmol) was added. The mixture was stirred at ambient temperature for 18 hours. After this, the solution was filtered through celite and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel, eluting with 30-100% EtOAc / hexane to give the title compound (34.76 g, 86%). ES / MS m / z 430.4 (m+H).

[0109] Preparation 3

[0110] 5-[3-Acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid

[0111]

[0112] A solution of methyl (5-acetamido-3,4-diacetoxy-6-hexyl-5-phenoxy-tetrahydropyran-2-yl)acetate (34.76 g, 80.93 mmol) in MeCN (174 mL) and DCM (174 mL) was cooled to 0°C. Sodium periodate solution (22.4 g, 104.7 mmol) was added, and stirring was continued at 0°C for 10 minutes. After this, ruthenium (III) chloride (270 mg, 1.3 mmol) was added, and the mixture was stirred while warming to ambient temperature. After stirring for 2 hours, additional sodium periodate (66 g, 308.4 mmol) was added, and stirring was continued for 18 hours. After this, the mixture was extracted with 3:1 CHCl:IPA (2 x 500 mL), washed with saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 0-40% MeOH / DCM to afford the title compound (29.75 g, 82%). ES / MS m / z 448.4 (m+H).

[0113] Preparation 4

[0114] Benzyl 6-aminohexanoate hydrochloride

[0115]

[0116] To a suspension of 6-aminohexanoic acid (5.00 g, 38.1 mmol) in THF (38 mL) was added benzyl alcohol (47 mL, 453.7 mmol), and the mixture was cooled to 0°C. Thionyl chloride (8.6 mL, 120 mmol) was added dropwise, and the mixture was stirred for 18 hours while warming to ambient temperature. After this time, diethyl ether (166 mL) was added, and the reaction vessel was transferred to a -20°C freezer for 1 hour. After this time, the solid precipitate was collected by filtration to provide the title compound (8.57 g, 81%). ES / MS m / z 222 (m+H).

[0117] Preparation 5

[0118] 11-Aminoundecanoic acid benzyl ester hydrochloride

[0119]

[0120] The title compound was prepared from 11-aminoundecanoic acid in a manner substantially analogous to Preparation 4. ES / MS m / z 292.2 (m+H).

[0121] Preparation 6

[0122] Allyl 11-aminoundecanoate hydrochloride

[0123]

[0124] A vessel was charged with 11-aminoundecanoic acid (9.00 g, 44.7 mmol) in allyl alcohol (42 mL) and the mixture was cooled to 0°C. Thionyl chloride (6.5 mL, 89.4 mmol) was added and the mixture was stirred for 18 hours while warming to ambient temperature. After this time, the mixture was concentrated in vacuo and diethyl ether (200 mL) was added to the residue to give a white suspension. The mixture was stirred at ambient temperature for 10 minutes and the solid precipitate was collected by filtration to give the product (12.0 g, 97%). ES / MS m / z 242.2 (m+H).

[0125] Preparation 7

[0126] (2S)-3-[Bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluorene-9-methoxycarbonylamino)propanoic acid

[0127]

[0128] Under 0 ℃ inert atmosphere, to (2S)-2-(9H-fluorenes-9-methoxycarbonylamino)-3-hydroxypropionic acid (40g, 0.122mol) in dry DCM (400mL) stirred solution, add DIEA (64mL, 0.366mol).To it, slowly add DMTCl (49.6g, 0.146mol) in DCM (200mL) solution.Gained reaction mixture is heated to ambient temperature and stirred 16 hours.After this, reaction mixture is diluted with water (12.5 volumes), and extracted with DCM (25 volumes).Organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo.With 10%EtOAc / hexane (12.5 volumes) washing the crude product obtained, and dry under vacuum, obtain light brown solid title compound (62g, crude product).This material is taken to next step without being further purified. Thin layer chromatography: 5% methanol / dichloromethane (Rf: 0.5) UV spectrophotometry, 254 nM.

[0129] Preparation 8

[0130] 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxo-ethyl]carbamate

[0131]

[0132] Under an inert atmosphere, to a stirred solution of (2S)-3-[bis(4-methoxyphenyl)phenylmethoxy]-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (62 g, 0.103 mol) in DCM (750 mL) at 0°C was slowly added HBTU (78.3 g, 0.206 mol), HOBt (27.9 g, 0.206 mol), and piperidin-4-ylmethanol (15.4 g, 0.134 mol), followed by TMP (15 mL, 0.113 mol). The resulting reaction mixture was allowed to warm to room temperature and stirred for 4 hours. After 4 hours, the reaction mixture was diluted with water (8 volumes) and extracted with DCM (15 volumes). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 20-40% EtOAc / hexanes and 1% MeOH / DCM to afford the title compound (40 g, 52% yield over two steps). 1 H NMR(DMSO-d6)δ7.88(br d,J=7.5Hz,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,J=11.4Hz,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).

[0133] Preparation 9

[0134] (2S)-2-Amino-3-[bis(4-methoxyphenyl)-phenylmethoxy]-1-[4-(hydroxymethyl)-1-piperidinyl]propan-1-one

[0135]

[0136] To 9H-fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxoethyl]carbamate (40 g, 0.055 mol) was slowly added a 20% piperidine solution in DMF (400 mL) at 0°C under an inert atmosphere. The reaction mixture was stirred at ambient temperature for 1 hour. After this time, the mixture was diluted with water (15 ml) and extracted with EtOAc (30 ml). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 1-8% MeOH / DCM to give the title compound (13 g, 47%) as an off-white solid. ES / MS m / z 1009.5 (2M+H).

[0137] Preparation 10

[0138] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid methyl ester

[0139]

[0140] To a flask containing (S)-4-((tert-Butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (7.00 g, 26.8 mmol) and HOBt (4.16 g, 30.8 mmol) were added DMF (179 mL) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (11.7 g, 30.9 mmol). DIEA (14 mL, 80.3 mmol) was added and the mixture was stirred at room temperature. The mixture was stirred for 5 minutes. Tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate (8.94 g, 29.5 mmol) was then added in one portion and stirring continued at room temperature. After stirring for 18 hours, the mixture was diluted with EtOAc (400 mL), washed with water (2 x 400 mL) and saturated aqueous sodium chloride (400 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel, eluting with 40-100% EtOAc / hexanes, to afford the title compound (13.01 g, 89%). ES / MS m / z 547.40 (M+H).

[0141] Preparation 11

[0142] (2S)-5-[Bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid

[0143]

[0144] A flask was charged with (2S)-methyl 5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoate (13.01 g, 23.8 mmol), tetrahydrofuran (THF) (120 mL), and methanol (MeOH) (120 mL). 1N sodium hydroxide (NaOH) (71 mL, 71 mmol) was added, and the mixture was stirred at room temperature. After 1 hour, the mixture was concentrated in vacuo and redissolved in water (300 mL). 5N hydrochloric acid (HCl) (12 mL) was added to lower the pH to 4. The mixture was extracted with dichloromethane (DCM) (3 x 300 mL), and the combined organic layers were washed with saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated to yield the title compound (12.41 g, 98%). ES / MS m / z 531.60 (MH).

[0145] Preparation 12

[0146] Allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoyl]amino]undecanoate

[0147]

[0148] To a flask containing (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (500 mg, 0.94 mmol) and 11-aminoundecanoic acid allyl ester hydrochloride (313 mg, 1.13 mmol) was added DMF (6.25 mL) and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (428 mg, To the mixture was added DIEA (0.5 mL, 3 mmol), and the mixture was stirred at room temperature for 18 hours. After stirring, the mixture was diluted with EtOAc (200 mL), washed with water (3 × 200 mL) and saturated aqueous sodium chloride (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel using 40-100% EtOAc / hexane as the eluent to afford the title compound (687 mg, 97%). 1H NMR(DMSO-d6)δ7.78-7.64(m,1H),6.98-6.7(m,2H),5.96-5.84(m,1H),5.31-5.25(m,1H),5.23-5.17(m,1H),4.56-4.50(m,2H),3.88-3.67(m ,1H),3.30-3.19(m,4H),3.11-2.91(m,6H),2.35-2.12(m,4H),1.88-1 .65(m,2H),1.58-1.47(m,2H),1.46-1.30(m,30H),1.30-1.18(m,12H).

[0149] Preparation 13

[0150] (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)undecanoic acid allyl ester

[0151]

[0152] To a solution of allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoate (687 mg, 0.91 mmol) in dichloromethane (DCM) (15 mL) was added trifluoroacetic acid (TFA) (15 mL). The mixture was stirred at room temperature. After 1.5 hours, the mixture was concentrated in vacuo. The residue was dissolved in methanol (MeOH) and placed on an ion exchange column. The ion exchange column was eluted first with methanol (150 mL) and then with 7N ammonia / methanol (150 mL). The basic fraction was concentrated in vacuo to give the title compound (410 mg, 99%). ES / MS m / z 456.4 (M+H).

[0153] Preparation 14

[0154] 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxopentanamido]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetylmethyl)tetrahydropyran-2]oxopentanamido]ethyl]amino]-5-oxopentanamido]undecanoic acid allyl ester

[0155]

[0156] A flask was charged with 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid (489 mg, 1.09 mmol) and allyl (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamidyl)undecanoate (150 mg, 0.33 mmol). DCM (3.35 mL) was added, followed by 1-hydroxybenzotriazole monohydrate (164 mg, 1.07 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (206 mg, 1.07 mmol). The mixture was stirred at room temperature for 18 hours. After stirring, the solution was diluted with EtOAc (100 mL) and washed with saturated NaHCO₃ (2×100 mL), saturated aqueous NH₄Cl (100 mL), and saturated aqueous sodium chloride (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel using 0-10% MeOH / DCM as eluent to afford the title compound (424 mg, 74%). ES / MS m / z 872.80 (M+2H) / 2.

[0157] Preparation 15

[0158] 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxopentanamido]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetylmethyl)tetrahydropyran-2]oxopentanamido]ethyl]amino]-5-oxopentanamido]undecanoic acid

[0159]

[0160] To allyl 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanamido]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanamido]ethyl]amino]-5-oxopentanamido]undecanoate (354 mg, 0.20 mmol) in dichloromethane (DCM) (2 mL) was added tetrakis(triphenylphosphine)palladium (29 mg, 0.02 mmol) followed by PhSiH3 (51 μL, 0.41 mmol). The mixture was stirred at room temperature for 2 hours and then diluted with saturated aqueous sodium bicarbonate solution (100 mL). 1N sodium hydroxide (15 mL) was added to bring the pH to approximately 10. The aqueous solution was washed with dichloromethane (DCM) (3 x 100 mL) and then acidified with concentrated hydrochloric acid (5 mL) and 5N aqueous hydrochloric acid (15 mL). The aqueous layer was extracted with dichloromethane (DCM) (100 mL), and the organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel using 0-20% methanol / dichloromethane to afford the title compound (151 mg, 44%). ES / MS m / z 852.60 (M+2H) / 2.

[0161] Preparation 16

[0162] (2,5-Dioxopyrrolidin-1-yl)11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxopentanamido]-5-[bis[2-[5-[3-acetylamino-4,5-diacetyloxy-6-(ethoxymethyl)tetrahydropyran-2-yl]oxopentanamido]-5-[bis[2-[5-[3-acetylamino-4,5-diacetyloxy-6-(ethoxymethyl)tetrahydropyran-2-yl]oxopentanamido]-

[0163]

[0164] To a reaction flask was added 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanamido]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanamido]ethyl]amino]-5-oxo-pentanamido]amino]undecanoic acid (50 mg, 0.03 mmol), N-hydroxysuccinimide (5 mg, 0.04 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8 mg, 0.04 mmol). DCM (0.3 mL) was added, and the mixture was stirred at room temperature. After 18 hours, the mixture was loaded directly onto a silica gel column and the crude mixture was purified by flash chromatography on silica gel eluting with 0-10% MeOH / DCM to afford the title compound (49 mg, 93%). ES / MS m / z 901.40 (M+2H) / 2.

[0165] Preparation 17

[0166] Benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoyl]amino]hexanoate

[0167]

[0168] The title compound was prepared from (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid and benzyl 6-aminohexanoate hydrochloride in a manner essentially analogous to the method for preparing 10. ES / MS m / z 736.40 (M+H).

[0169] Preparation 18

[0170] Benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxopentanoyl]amino]hexanoate tris(trifluoroacetic acid)

[0171]

[0172] To a solution of benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoate (15.47 g, 21.02 mmol) in DCM (105 mL) was added TFA (16 mL, 210.2 mmol). The mixture was stirred at ambient temperature for 24 hours. Thereafter, additional TFA (16 mL, 210.2 mmol) was added and stirring was continued for an additional 2 hours. Thereafter, the mixture was concentrated in vacuo. The resulting residue was azeotroped with toluene (2×30 mL). The resulting oil was further dried in a vacuum oven at 40° C. for 4 hours to give the title compound (28.08 g, 58% purity (based on residual toluene), 99+%). ES / MS m / z 436.40 (M+H). This compound was dissolved in 70 mL of DMF to make a 0.3 M solution for the next step.

[0173] Preparation 19

[0174] Benzyl 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0175]

[0176] The title compound was prepared from 5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid and benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoate trifluoroacetic acid in a manner substantially analogous to the method for Preparation 10. ES / MS m / z 862 (M+2H) / 2.

[0177] Preparation 20

[0178] 6-[[(2S)-2-[5-[3-Acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid

[0179]

[0180] Palladium on carbon (1.90 g, 0.89 mmol, 5% by mass, 50% humidity) is placed in a round-bottom flask, and the container is evacuated and back-flushed three times with nitrogen. A solution of 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]benzyl hexanoate (15.41 g, 8.94 mmol) in MeOH (178 mL) is added via a syringe. The flask is evacuated and back-flushed with 1 atm of hydrogen, and the mixture is stirred at room temperature under 1 atm of hydrogen for 18 hours. After this time, the mixture was filtered through celite and the filtrate was concentrated in vacuo to give the title compound (13.85 g, 95%). ES / MS m / z 817.2 (M+2H) / 2.

[0181] Preparation 21

[0182] (2,5-Dioxopyrrolidin-1-yl)6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0183]

[0184] The title compound was prepared from 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid in a manner substantially similar to that of Preparation 16. ES / MS m / z 866.20 (M+2H) / 2.

[0185] Preparation 22

[0186] (2S)-5-[Bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid benzyl ester

[0187]

[0188] The title compound was prepared from tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate and (4S)-5-benzyloxy-4-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid in a manner essentially analogous to the method of Preparation 12. ES / MS m / z 623.6 (M+H).

[0189] Preparation 23

[0190] (2S)-2-Amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoic acid benzyl ester tris(trifluoroacetate)

[0191]

[0192] The title compound was prepared from benzyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate in a manner substantially analogous to that of Preparation 18. ES / MS m / z 323.2 (M+H).

[0193] Preparation 24

[0194] (2S)-5-[Bis[2-[5-(tert-Butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-Butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoic acid benzyl ester

[0195]

[0196] The title compound was prepared from 5-(tert-butoxycarbonylamino)pentanoic acid and (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoic acid benzyl ester tris(trifluoroacetate) in a manner substantially analogous to the method of Preparation 10. ES / MS m / z 920.6 (M+H).

[0197] Preparation 25

[0198] (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoic acid benzyl ester tris(trifluoroacetate)

[0199]

[0200] The title compound was prepared from (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoic acid benzyl ester in a manner substantially analogous to that of Preparation 18. ES / MS m / z 620.4 (M+H).

[0201] Preparation 26

[0202] (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid benzyl ester

[0203]

[0204] The title compound was prepared from 5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid and (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoic acid benzyl ester tri(trifluoroacetate) in a manner substantially similar to Preparation 10. ES / MS m / z 954.80 (M+2H) / 2.

[0205] Preparation 27

[0206] (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid

[0207]

[0208] Carbon-supported palladium (467 mg, 0.22 mmol, 5% by mass, 50% humidity) was placed in a round-bottom flask, and the flask was evacuated and back-flushed three times with nitrogen. A solution of (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid benzyl ester (4.19 g, 2.20 mmol) in MeOH (44 mL) was added via a syringe, followed by the addition of three drops of acetic acid. The flask was evacuated and back-flushed with 1 atm of hydrogen, and the mixture was stirred at room temperature under 1 atm of hydrogen. After 2 hours, the mixture was filtered through celite and the filtrate was concentrated in vacuo to give the title compound (3.99 g, 99+%). ES / MS m / z 909.6 (M+2H) / 2.

[0209] Preparation 28

[0210] Benzyl 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0211]

[0212] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 6-aminohexanoate hydrochloride in a manner substantially similar to that of Preparation 10. ES / MS m / z 1011.6 (M+2H) / 2.

[0213] Preparation 29

[0214] 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid

[0215]

[0216] Palladium on carbon (24 mg, 0.01 mmol, 5% by mass, 50% humidity) was placed in a round-bottom flask, and the flask was evacuated and back-flushed with nitrogen. A solution of 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]benzyl hexanoate (222 mg, 0.11 mmol) in MeOH (2.2 mL) was added via a syringe, followed by three drops of acetic acid. The flask was evacuated and back-flushed with 1 atm of hydrogen, and the mixture was stirred at room temperature under 1 atm of hydrogen. After 5 hours, the flask was purged with nitrogen and the mixture was filtered through celite. The filtrate was concentrated in vacuo to give the title compound (180 mg, 85%). ES / MS m / z 966.2 (M+2H) / 2.

[0217] Preparation 30

[0218] 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid (2,5-dioxopyrrolidin-1-yl) ester

[0219]

[0220] The title compound was prepared from 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid in a manner substantially similar to that of Preparation 16. ES / MS m / z 1014.6 (M+2H) / 2.

[0221] Preparation 31

[0222] Benzyl 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate

[0223]

[0224] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 11-aminodecanoate hydrochloride in a manner substantially similar to the method of Preparation 10. ES / MS m / z 1046.6 (M+2H) / 2.

[0225] Preparation 32

[0226] 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid

[0227]

[0228] To a round-bottom flask was added palladium on carbon (35 mg, 0.02 mmol, 5% by mass, 50% humidity), and the flask was evacuated and back-flushed with nitrogen three times. 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid benzyl ester (285 mg, 80% purity, 0.11 mmol) solution was added via a syringe. The container was evacuated and back-flushed with 1 atm of hydrogen, and the mixture was then stirred at room temperature under 1 atm of hydrogen. After stirring for 3 hours, the flask was purged with nitrogen, and the mixture was filtered through celite. The filtrate was concentrated to give the title compound (213 mg, 79% purity, 77%). ES / MS m / z 1001.20 (M+2H) / 2.

[0229] Preparation 33

[0230] 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid (2,5-dioxopyrrolidin-1-yl) ester

[0231]

[0232] The title compound was prepared from 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid in a manner substantially similar to that of Preparation 16. ES / MS m / z 1050 (M+2H) / 2

[0233] Preparation 34

[0234] [5-Acetylamino-6-[5-[2-[[(4S)-4-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxo-pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate

[0235]

[0236] The title compound was prepared from 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid and (2S)-2-amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1-piperidinyl]propan-1-one in a manner substantially similar to the method of Preparation 10. ES / MS m / z 1059.2 (M-2H) / 2.

[0237] Preparation 35

[0238] 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propionyl]-4-piperidinyl]methoxy]-4-oxobutanoic acid

[0239]

[0240] [5-Acetylamino-6-[5-[2-[[(4S)-4-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-ethyl To a solution of [4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxo-pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (1.194 g, 0.56 mmol) in DCM (11 mL) was added succinic anhydride (113 mg, 1.13 mmol), TEA (0.4 mL, 3 mmol), and DMAP (213 mg, 1.69 mmol). The mixture was stirred at ambient temperature for 1 hour. Thereafter, the mixture was diluted with saturated NH4Cl (200 mL) and extracted with DCM (3×200 mL) and 3:1 CHCl3:IPA (200 mL). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 0-40% MeOH / DCM and the product was dried in a vacuum oven at 40°C for 3 hours to give the title compound (1.081 g, 86%). ES / MS m / z 1109.60 (M-2H) / 2.

[0241] Preparation 36

[0242] Resin loading

[0243]

[0244] A solution of 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propionyl]-4-piperidinyl]methoxy]-4-oxo-butanoic acid (1.00 g, 0.61 mmol) in MeCN (6 mL) and DCM (1 mL) was transferred to the resin loading cartridge. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (386 mg, 0.97 mmol) and DIEA (0.25 mL, 0.48 mmol) were added to the container and shaken at room temperature for 5 minutes. LCAA controlled pore glass resin (5.39 g, 90 μmol / g loading, purchased from ChemGenes) was added and the mixture was shaken at room temperature for 18 hours. Afterwards, the cartridge was drained with suction and the resin was washed with DCM (10 mL) by shaking for 10 minutes. The cartridge was drained and the washing and draining steps were repeated with 10% MeOH / DCM (10 mL) and Et2O (10 mL). After draining, a solution of acetic anhydride (6.4 mL), pyridine (20 mL), and TEA (0.22 mL) was added and the cartridge was shaken for 2 hours. Afterwards, the cartridge was drained and the washing and draining steps were repeated with DCM (10 mL), 10% MeOH / DCM (10 mL), and ether (10 mL). After draining, the resin was vacuum dried for 30 minutes. The resin loading was determined using standard trityl analysis. The calculated resin loading was 34.7 μmol / g.

[0245] Preparation 37

[0246] Benzyl 2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetate

[0247]

[0248] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 2-(2-aminoethoxy)acetate hydrochloride in a manner substantially similar to that of Preparation 10. ES / MS m / z 1005.2 (M+2H / 2).

[0249] Preparation 38

[0250] 2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]acetic acid

[0251]

[0252]

[0114] Benzyl 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetate (0.120 mmol, 240 mg) was combined with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 ml). The mixture was hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 48 minutes, filtered through celite and concentrated in vacuo to give the title compound (187 mg, 82%) as a grey solid. ES / MS m / z 960.0 (M+2H / 2).

[0253] Preparation 39

[0254] (2,3,5,6-Tetrafluorophenyl) 2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetate

[0255]

[0256] To a mixture of 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetic acid (0.096 mmol, 184 mg) and DIEA (0.765 mmol, 140 μL) in DCM (3.0 ml) was added 2,2,2-trifluoroacetate (0.383 mmol, 100 mg) dropwise. The mixture was stirred at ambient temperature for 16 hours. The reaction mixture was directly purified by flash chromatography on silica gel eluting with 0% to 50% MeOH / DCM to afford the title compound as a tan solid (197 mg, 99%). ES / MS m / z 1034.0 (M+2H / 2).

[0257] Preparation 40

[0258] Benzyl 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetate

[0259]

[0260] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetate hydrochloride in a manner essentially analogous to the method of Preparation 10. ES / MS m / z 1049.0 (M+2H / 2).

[0261] Preparation 41

[0262] 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid

[0263]

[0264] Benzyl 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetate (0.118 mmol, 247 mg) was combined with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 mL). The mixture was hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 1 hour, filtered through celite and concentrated in vacuo to give the title compound (227 mg, 96%) as a grey solid. ES / MS m / z 1004.0 (M+2H / 2).

[0265] Preparation 42

[0266] (2,3,5,6-Tetrafluorophenyl) 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetate

[0267]

[0268] To a mixture of 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (0.111 mmol, 222 mg) and DIEA (0.883 mmol, 154 μL) in DCM (3.0 ml) was added 2,3,5,6-tetrafluorophenyl) 2,2,2-trifluoroacetate (0.443 mmol, 116 mg) dropwise. The mixture was stirred at ambient temperature for 16 hours. The reaction mixture was directly purified by flash chromatography on silica gel eluting with 0% to 50% MeOH / DCM to afford the title compound as a tan solid (174 mg, 73%). ES / MS m / z 1078.2 (M+2H / 2).

[0269] Example 1: Conjugation protocol

[0270] To synthesize the GalNAc-conjugated sense strand, 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 equivalent) was prepared. Borate buffer (10% v / v; 20x) was added to the oligonucleotide C6-NH2 sense strand in an Eppendorf tube, followed by the addition of the GalNAc ligand (5 equivalents). The mixture was shaken at room temperature for 16 hours. Thereafter, the mixture was transferred to a 15 mL Falcon tube, ammonium hydroxide (28% by mass) was added, and shaken at room 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 / 20 mM NaH2PO4, 1 M NaBr; 35% to 55% B over 5 CV at 8 mL / min, column temperature 60°C. The desired fractions were collected and desalted by spin filtration using an Eppendorf centrifuge or desalting columns. After desalting, the product was recovered and the OD and volume were measured to obtain the concentration.

[0271] Alternatively, conjugation to the 5' position of the sense strand can be achieved by immobilizing the GalNAc ligand on microporous polystyrene resin or controlled pore glass and synthesizing it with 5'-CE (β-cyanoethyl) phosphoramidite using established solid-phase oligonucleotide synthesis methods.

[0272] Alternatively, the GalNAc ligand is converted to a suitable phosphoramidite and delivered to the 5' position of the sense strand using standard phosphoramidite chemistry.

[0273] Example 2: Annealing

[0274] To generate siRNA duplexes of sense and antisense strands, the following procedure was performed: the corresponding antisense oligonucleotide (1 equivalent) was added to a Falcon tube containing the oligonucleotide sense strand-GalNAc conjugate and vortexed for 10 seconds, then spin filtered through a 100K MWCO Amicon filter unit to remove particulate matter. The filtrate was recovered and vacuum concentrated on a Genevac evaporator. The residue was reconstituted in 1xPBS, filtered through a 0.2μ filter, and the OD and volume were measured to obtain the concentration.

[0275] use Endotoxin testing was performed using Limulus Amebocyte Lysate on the Nexgen PTS instrument.

[0276] Table 1 - Exemplary molecules synthesized using the above conjugation and annealing protocol.

[0277]

[0278]

[0279] Example 3:

[0280] General procedure for oligonucleotide synthesis using GalNAc-functionalized CPG

[0281] Oligonucleotide synthesis at MerMade TM 12 instrument using phosphoramidite chemistry. The sense strand was synthesized on a pre-functionalized GalNAc solid support, while the antisense strand was synthesized using a standard support preloaded with the first nucleotide of the oligonucleotide sequence. The oligonucleotides were cleaved and deprotected with concentrated ammonium hydroxide solution (28% by mass) and purified by ion exchange chromatography using the conditions described above. Desalting, annealing, and endotoxin testing were performed.

[0282] Antisense oligonucleotide sequences were designed using 15 to 50 nucleotides of the antisense strand described herein, including those in Tables 2A, 2B and Tables 3A, 3B.

[0283] Tables 2A and 2B below show exemplary antisense strand sequences of 23 nucleotides in length, which can optionally be further modified and synthesized and incorporated into RNAi agents as described herein.

[0284] Table 2A: Modified ANGPTL8 sequences for GalNAc-RNAi reagents

[0285]

[0286] Table 2B: Modified ANGPTL8 sequences for GalNAc-RNAi reagents

[0287]

[0288]

[0289] Table 3A: Modified sense and antisense strands for GalNAc-ANPTL8

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297] P or [Phos] indicates 5' phosphate

[0298] m indicates 2'O-methyl modified ribose sugar on the listed nucleotides

[0299] f indicates a 2'F modified ribose sugar on the listed nucleotides

[0300] * indicates a phosphorothioate bond (instead of a phosphodiester bond)

[0301] Table 3B: Modified sense and antisense strands of the GalNAc-ANPTL8 RNAi reagents used herein

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] m indicates 2'O-methyl modified ribose sugar on the listed nucleotides

[0310] f indicates a 2'F modified ribose sugar on the listed nucleotides

[0311] * indicates a phosphorothioate bond (not a phosphodiester bond)

[0312] Table 4: Sense and antisense strands of GalNAc-ANPTL8

[0313]

[0314]

[0315]

[0316]

[0317] Human ANGPTL8 transcript NM_018687.7 SEQ ID 511:

[0318]

[0319] Example 4: Administration of GalNAc-conjugated ANGPTL8 siRNA in primary liver cells of AAV-ANGPTL8 humanized mice In vitro knockdown of human ANGPTL8 in MPH cells

[0320] Knockdown of ANGPTL8 expression by LYGal1-conjugated ANGPTL8 siRNA was determined using the following procedures: Mouse primary hepatocytes (MPH) were freshly isolated from AAV-ANGPTL8 humanized mice, plated at 15,000 cells per well in a Corning 96-well plate, and siRNA was added directly to the wells. For single-point (SP) screening, 1 μM (1,000 nM) of GalNAc-conjugated siRNA was used. To generate a concentration / dose response curve, 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 were used. Treated cells were lysed, and RNA was isolated directly into a 96-well plate using the Quick-RNA 96 Kit (Zymo Research). Eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and the following steps in a thermal cycler: 37° C. for 30 minutes, 95° C. for 5 minutes, and hold at 4° C. Polymerase chain reaction (PCR) was performed by TaqMan RTPCR (Life Technologies) using the following cycle 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.

[0321] Human ANGPTL8 levels were normalized to mouse Rplp0 (Life Technologies) and expressed as relative knockdown of human ANGPTL8 mRNA expression compared to vehicle-treated control cells. IC values were calculated using XLFit with a four-parameter fitting model. 50 value.

[0322] Table 5 shows the results of a single-dose screen in AAV-ANGPTL8 humanized mouse primary hepatocytes using free uptake of the indicated GalNAc-conjugated ANGPTL8 siRNAs. Data are presented as percentage of message knockdown relative to untreated cells. Also included are the IC values of the top hits from the single-point screen. 50 and maximum knockdown percentage, and subsequent concentration / dose-response curve data.

[0323] Table 5: Percent knockdown in single-dose screening and IC of top hits from single-point screening in AAV-ANGPTL8 humanized mouse primary hepatocytes 50 and maximum knockdown percentage.

[0324]

[0325]

[0326]

[0327]

[0328] Example 5: In vitro knockdown of human ANGPTL8 in Hep3B cells using GalNAc-conjugated ANGPTL8 siRNA

[0329] The knockdown of ANGPTL8 expression by LYGal1-conjugated ANGPTL8 siRNA was determined using the following procedure: 0.3 μl / well of transfection reagent RNAiMAX (Life Technologies) was mixed with siRNA in a Corning 96-well plate, and then 8,000 Hep3B cells (ATCC) were added per well. To generate a concentration / dose response curve, GalNAc-conjugated siRNA 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 were used.

[0330] The treated cells were lysed and RNA was isolated directly into 96-well plates using the Quick-RNA 96 kit (Zymo Research). The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and the following steps in a thermal cycler: 37°C for 30 minutes, 95°C for 5 minutes, and maintained at 4°C. Polymerase chain reaction (PCR) was performed using TaqMan RT PCR (Life Technologies) using the following cycle 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.

[0331] Human ANGPTL8 levels were normalized to human RPLP0 (Life Technologies) and expressed as relative knockdown of human ANGPTL8 mRNA expression compared to vehicle-treated control cells. IC values were calculated using XLFit with a four-parameter fitting model. 50 value.

[0332] Table 6 shows the IC calculated from the concentration / dose response curves in Hep3B cells by transfection of the indicated ANGPTL8 siRNAs. 50In most cases, the duplex resulted in more than 90% knockdown, and the IC 50 At nanomolar levels or lower.

[0333] Table 6: IC of ANGPTL8 messenger in Hep3B cells by transfection with RNAiMAX reagent and the indicated ANGPTL8 siRNAs 50 and maximum knockdown percentage.

[0334]

[0335]

[0336] Example 6: In vivo single-dose screening

[0337] GalNAc-siRNA was tested in male C57bl / 6 mice (Taconic farms) (n=16). These siRNAs were tested in a single study. Mice were administered an adeno-associated virus (AAV) vector (Vector BioLabs) containing a plasmid carrying the albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7) via retro-orbital injection. Blood was collected from mice via the retro-orbital sinus 14 days after AAV administration. Serum was prepared from the blood, and triglycerides were measured using a COBAS clinical chemistry analyzer (Roche), and ANGPTL4 / 8 was measured by ELISA (Meso Scale Diagnostics). The body weight of the mice was measured 22 days after AAV administration. Mice with similar body weight, serum triglyceride levels, and serum ANGPTL4 / 8 were assigned to groups (n=6 / group). Mice were subcutaneously administered PBS or a 5 mg / kg dose of the GalNac-siRNA test article. 7 days after subcutaneous injection, blood of all mice was collected and serum triglyceride was analyzed. 14 days after subcutaneous injection, mice were euthanized under isoflurane anesthesia, blood was collected and serum triglyceride was analyzed. Liver was collected from mice and frozen in liquid nitrogen. The percentage change of triglyceride relative to the time-matched PBS was calculated: [(triglyceride-triglyceride of PBS group) / triglyceride value of PBS group]*100. Using the Lysing Matrix D bead tube on FastPrep-24 (MP Bio), the liver was homogenized in TriZol (Invitrogen). Chloroform was added, and the aqueous phase was mixed with ethanol to precipitate RNA. According to the manufacturer's protocol, RNA was isolated on the column using PureLink Pro96 total RNA purification kit (Invitrogen) and quantified on NanoDrop (ThermoFisher). Equal amounts of RNA (1 μg) were reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) on a Mastercycler Nexus (Eppendorf). The thermal cycler was set at 25°C for 10 minutes, 37°C for 2 hours, and then 85°C for 5 minutes. Template cDNA was mixed with Taqman Universal Master Mix and ready-to-use primer / probe sets, and RT-PCR was performed on a QuantStudio Pro7 (ThermoFisher) using the following parameters: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fold change (FC) was calculated by subtracting the CT value of mouse Rplp0 from the CT value of human ANGPTL8 to obtain the ΔCT value.The ΔΔCT value of the untreated sample (average of the PBS control) was then subtracted from the ΔCT value of each test sample to calculate the ΔΔCT value. The fold change was calculated by taking the logarithm to the base 2 of the negative ΔΔCT value. The percentage of mRNA remaining was calculated by multiplying the fold change by 100. The data are shown in Table 7.

[0338] Table 7: In vivo single-dose screening of hANGPTL8 AAV in C57 mice

[0339]

[0340]

[0341] Example 7: 8-week in vivo persistence

[0342] GalNAc-siRNA (n=11) was tested in male transgenic mice (Taconicfarms) targeting human cholesteryl ester transfer protein (CETP) and human apolipoprotein A1. siRNA was divided into two studies (n=7 and n=4) and tested. Mice were administered two adeno-associated viral (AAV) vectors via retro-orbital injection. One vector contained a plasmid (Vector BioLabs) with the albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7). The second vector contained the mouse codon-optimized sequence for human ANGPTL3 (NP_055310.1) (Vector BioLabs). Blood was collected from mice 3-4 weeks after AAV administration. This was considered a baseline blood collection. Serum was prepared from the blood, and ANGPTL3 / 8 was measured by ELISA (Meso Scale Diagnostics), and triglycerides were measured as described. The mice were weighed and assigned to groups with similar body weight, serum triglyceride, and ANGPTL3 / 8 levels (n=9 / group). 4-5 weeks after AAV administration, mice were subcutaneously administered PBS or 1.75 and 10 mg / kg doses of the test substance, GalNac-siRNA. 2 weeks after siRNA administration, 3 mice in each group were euthanized under isoflurane anesthesia, and blood was collected and serum triglycerides were analyzed. The livers were collected from the mice and frozen in liquid nitrogen. 2, 4, and 6 weeks after siRNA administration, blood was collected from the remaining mice under isoflurane anesthesia. Serum was prepared from the blood and triglycerides were measured. 8 weeks after siRNA administration, the remaining mice were euthanized under isoflurane anesthesia. Blood and livers were collected from the mice. The livers were processed and the remaining mRNA was calculated as described in the in vivo single-dose screen. The percent change in triglycerides relative to PBS was calculated as described in the in vivo single-dose screen.

[0343] Table 8: In vivo persistence, 8-week dose response (hANGPTL8 AAV in C57 mice)

[0344]

[0345] Example 8: 15-week in vivo persistence (dose response (C57 mouse hANGPTL8 AAV)) GalNAc-siRNA was tested in male transgenic mice (Taconic farms) against human cholesteryl ester transfer protein (CETP) and human apolipoprotein A1 (n=5). siRNA was tested in a single study. Mice were administered two adeno-associated viral (AAV) vectors by retro-orbital injection. One vector contained a plasmid (Vector BioLabs) with the albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7). The second vector contained the mouse codon-optimized sequence for human ANGPTL3 (NP_055310.1) (Vector BioLabs). Blood was collected from mice 3 weeks after AAV administration. This was considered a baseline blood collection. Serum was prepared from the blood, and triglycerides and ANGPTL3 / 8 were measured. Mouse body weight was measured, and mice with similar body weight, serum triglyceride, and ANGPTL3 / 8 levels were assigned to groups (n=10 / group). Four weeks after AAV administration, mice were subcutaneously administered PBS or 0.3, 1.75, and 10 mg / kg doses of the test substance GalNac-siRNA. Two weeks after siRNA administration, three mice in each group were euthanized under isoflurane anesthesia, blood was collected, and serum triglycerides were analyzed. Liver was collected from mice and frozen in liquid nitrogen. 3, 6, 9, and 12 weeks after siRNA administration, blood was collected from the remaining mice (n=7 / group) under isoflurane anesthesia. Serum was prepared from the blood and triglycerides were measured. 15 weeks after siRNA administration, the remaining mice were euthanized under isoflurane anesthesia. Blood and liver were collected from mice. Serum was prepared from the blood and triglycerides were measured. The liver was processed and the remaining mRNA was calculated as described in the in vivo single-dose screening. The percentage change in triglycerides relative to PBS was calculated as described in the in vivo single-dose screening.

[0346] Table 9: Dose Response (C57 Mouse hANGPTL8 AAV)

[0347]

Claims

1. An RNAi agent for reducing expression of an ANGPTL8 gene, wherein the RNAi agent comprises a delivery moiety of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand: wherein R is conjugated to the attachment point E of Formula I, 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 NO: 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or a sequence with 90% sequence identity thereof, or the antisense strand sequence shown in Tables 3A, 3B and 4, or a sequence with 90% sequence identity thereof, and wherein the sense strand and antisense strand each optionally comprise one or more modified nucleotides and optionally one or more linkages between the modified nucleotides.

2. The RNAi agent of claim 1, wherein Formula I is conjugated to the sense strand, optionally via a linker.

3. The RNAi agent of claim 2, wherein Formula I is conjugated to the 3' terminal nucleotide of the sense strand, optionally via a linker.

4. The RNAi agent of any one of claims 1 to 3, wherein the sense strand is 15 to 25 nucleotides in length.

5. The RNAi agent of any one of claims 1 to 4, wherein the sense strand is 18 to 23 nucleotides in length.

6. The RNAi agent of any one of claims 1-5, wherein the sense strand is 18-21 nucleotides in length.

7. The RNAi agent of any one of claims 1-6, wherein the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

8. The RNAi agent of any one of claims 1-7, wherein the sense strand and the antisense strand form a complementary region of at least 18 nucleotides in length.

9. The RNAi agent of any one of claims 1-8, 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.

10. The RNAi agent of any one of claims 1-9, wherein the duplex region between the sense strand and the antisense strand comprises 0 mismatches between the sense strand and the antisense strand.

11. The RNAi agent of any one of claims 1-10, wherein the antisense strand comprises 15 consecutive nucleotides of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or any one of the sequences of Tables 3A, 3B, and 4.

12. The RNAi agent of any one of claims 1-11, wherein the antisense strand comprises 18 consecutive nucleotides of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or any one of the sequences of Tables 3A, 3B, and 4.

13. The RNAi agent of any one of claims 1-12, wherein the sense strand is selected from SEQ ID NOs: 1-5 or a sequence having at least 90% sequence identity thereto, or a sense strand sequence shown in Table 3 or a sequence having at least 90% sequence identity thereto.

14. The RNAi agent of any one of claims 1-13, wherein the sense strand or the antisense strand each independently comprises one or more modified nucleotides.

15. The RNAi agent of any one of claims 1 to 14, wherein each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide, and the modified nucleotides are independently 2'-fluoro-modified nucleotide residues or 2'-O-methyl-modified nucleotides.

16. The RNAi agent of any one of claims 1 to 15, wherein the antisense strand is 23 nucleotides in length, and each nucleotide of the antisense strand is a modified nucleotide, and the position of the 2'-fluoro modified nucleotide is modified to be present in a group selected from the group consisting of: 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.

17. The RNAi agent of any one of claims 1-16, wherein the sense strand and antisense strand each independently comprise one or more modified internucleotide linkages, and wherein each modified internucleotide linkage is a phosphorothioate linkage.

18. The RNAi agent of any one of claims 1-17, wherein the sense strand and antisense strand each independently comprise four phosphorothioate linkages.

19. The RNAi agent of any one of claims 1 to 18, wherein the 5'-terminal nucleotide of the antisense strand comprises an OH group, a phosphate group, a vinylphosphonate, or a phosphate analog.

20. The RNAi agent of claim 19, wherein the 5'-terminal nucleotide of the antisense strand is further modified by replacing the 5'-phosphate group with an OH group.

21. The RNAi agent of any one of claims 1-20, wherein R is conjugated to Formula I via a linker.

22. The RNAi agent of any one of claims 1-21, wherein R is conjugated to Formula I via a linker, and wherein the linker comprises a linker of Formula II having attachment points A and B, or a linker of Formula III having attachment points C and D, and wherein: a. Formula I is conjugated to attachment point A of Formula II at attachment point E, and Formula II is conjugated to a phosphate group or a phosphorothioate group at attachment point B, and the phosphate group or the phosphorothioate group is further conjugated to R; b. Formula I is conjugated to attachment point C of Formula III at attachment point E, and Formula III is conjugated to a phosphate group or a phosphorothioate group at attachment point D, and the phosphate group or the phosphorothioate group is further conjugated to R.

23. The RNAi agent of any one of claims 1-22, wherein R is conjugated to Formula I via a linker, and wherein the linker is a linker of Formula III comprising attachment points C and D: And wherein Formula I is conjugated to attachment point C of Formula III at attachment point E, and Formula III is conjugated to a phosphate group or a phosphorothioate group at attachment point D, and the phosphate group or the phosphorothioate group is further conjugated to R.

24. The RNAi agent of any one of claims 1-23, wherein the RNA interference agent reduces expression of the ANGPTL8 gene in hepatocytes compared to a control agent.

25. The RNAi agent of any one of claims 1-24, for use in therapy.

26. The RNAi agent of any one of claims 1-24, for use in treating a disease or condition selected from the group consisting of cardiovascular disease, cardiometabolic disease, diabetes, dyslipidemia, renal dysfunction, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), and obesity.

27. The RNAi agent for use according to claim 26, wherein the disease or pathology is dyslipidemia.

28. A pharmaceutical composition comprising the RNAi agent of any one of claims 1-24, and one or more pharmaceutically acceptable excipients.

29. Use of the RNAi agent of any one of claims 1 to 24 in the preparation of a medicament for treating a disease or condition selected from the group consisting of cardiovascular disease, diabetes, dyslipidemia, abnormal renal function, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), and obesity.

30. The use according to claim 29, wherein the disease or disorder is dyslipidemia.

31. A method of treating dyslipidemia in a patient in need thereof, comprising administering to the patient the RNAi agent of any one of claims 1-24 or a pharmaceutical composition thereof.

32. A method of reducing ANGPTL8 expression in a cell, comprising contacting the cell with the RNAi agent of any one of claims 1-24.

33. The method of claim 32, wherein the method further comprises incubating the cells for a sufficient period of time to reduce ANGPTL8 mRNA levels by at least 50% compared to untreated or control-treated cells.

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