Novel FAS RNAi therapeutic agent and its use

Abstract

The present invention relates to novel therapeutic compounds known as RNAi agents that decrease the expression of the FAS receptor (expressed by the FAS gene), thereby decreasing FAS mRNA expression and protein expression. Such RNAi agents are useful in the treatment of diseases, such as autoimmune hepatitis, that involve the modulation of FAS expression and function.

Description

[Background technology]

[0001] The present invention relates to novel therapeutic compounds known as RNAi agents that decrease the expression of FAS (expressed by the FAS gene), thereby decreasing the expression of FAS mRNA and FAS protein. Such RNAi agents are useful in the treatment of diseases, such as autoimmune hepatitis, that involve the modulation of FAS expression and function.

[0002] FAS, the Fas death receptor, and its ligand, FASL, are members of the TNFR superfamily. Binding of FASL to FAS leads to downstream death-inducing signaling involving caspases (e.g., caspase 8 and 10) and the Fas-associated death domain protein (FADD) in a complex. Autoproteolysis of caspases within the complex initiates the caspase cascade, leading to apoptosis. NF-κB, MAPK3 / ERK1, and MAPK8 / JNK are also known to be activated by FAS signaling, and such activation is thought to result in proliferation in normal diploid fibroblasts and T cells. These play important roles in regulating immune responses involving FAS-expressing cells, including hepatocytes.

[0003] Autoimmune hepatitis (AIH) is a chronic inflammation of the liver without an identifiable cause, such as a viral infection. Patients with AIH have increased levels of FAS in hepatocytes. Genetics, environment (e.g., environmental triggers), and dysregulation of the innate immune system are thought to contribute to the progression of the disease from inflammation to liver fibrosis. AIH often first appears when patients reach their teenage years.

[0004] Treatment options are limited and often involve high-dose steroid therapy in combination with another immunosuppressant, azathioprine. Treatment correlates with downregulation of FAS, allowing patients to achieve near biochemical remission of inflammation. However, steroid therapy, like other immunosuppressant treatments such as azathioprine, can cause a wide range of serious side effects, especially when administered over a long period and / or at high doses. These can include the development of diabetes, bone thinning (osteoporosis), fractures (osteonecrosis), hypertension, cataracts, glaucoma, and weight gain. If treatment is discontinued, patients often experience relapse, and some experience disease progression requiring liver transplantation. Therefore, improved treatments for AIH are needed. Summary of the Invention

[0005] In one embodiment, an RNAi agent for reducing FAS gene expression, wherein the RNAi agent is a compound of formula I conjugated to R [ka] wherein R is double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand, and R is conjugated to connection point E of Formula I, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, the antisense strand comprises a region of complementarity to the FAS mRNA target sequence of SEQ ID NO: 1, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages. In some embodiments, Formula I is conjugated to the sense strand, optionally via a linker. In some embodiments, Formula I is conjugated to the 3'-terminal nucleotide of the sense strand, optionally via a linker.

[0006] 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.

[0007] In some embodiments, the sense strand or antisense strand comprises a sequence selected from Table 2, Table 3A, Table 3B, Table 4A, Table 4B, Table 7, or Table 8 disclosed herein. In some embodiments, the sense strand and antisense strand comprise a sequence selected from Table 2, Table 3A, Table 3B, Table 4A, Table 4B, Table 7, or Table 8 disclosed herein.

[0008] In some embodiments, R is conjugated to Formula I via a linker. In further embodiments, the linker is Formula II having attachment points A and B. [ka] or the linker comprises Formula III having attachment points C and D; a. Formula I is conjugated to Formula II at attachment point A, and Formula II is conjugated to a phosphate group at attachment point B, which is further conjugated to R; or b. Formula I is conjugated to Formula III at connection point C, and Formula III is conjugated to a phosphate group at connection point D, which is further conjugated to R.

[0009] In another aspect, provided herein is a pharmaceutical composition comprising a FAS RNAi agent described herein and one or more pharmaceutically acceptable excipients.

[0010] In another aspect, provided herein is a method of treating autoimmune hepatitis (AIH) in a patient in need thereof, comprising administering to the patient a FAS RNAi agent or pharmaceutical composition thereof described herein.

[0011] In another aspect, provided herein is a FAS RNAi agent for use in treatment.Also provided herein is a FAS RNAi agent for use in the treatment of AIH.Also provided herein is the use of a FAS RNAi agent in the manufacture of a medicament for the treatment of AIH. DETAILED DESCRIPTION OF THE INVENTION

[0012] Although FAS siRNA and ASO have been reported, none have been developed for patient treatment, including the treatment of AIH. The use of the FAS RNAi agents herein to reduce FAS expression can be used to treat AIH in patients in need thereof. Such siRNAs may exhibit one or more of the following: improved knockdown in the liver; improved tissue exposure; improved exposure in liver hepatocytes; improved durability response; improved pharmacokinetic profile; reduced off-target effects; and / or improved toxicity profile, compared to other liver-targeting siRNAs, such as FAS siRNAs containing different delivery ligands, different sequences, or different modified sequences, or compared to treatment with a vehicle control. Other embodiments of the FAS RNAi agents herein may include one or more of the following: reduced side effects; improved toxicity profile; improved safety profile; improved tolerability or compliance; and / or improved liver function tests, compared to steroids or other standard treatments. Still other siRNAs herein may have other advantages, including, for example, improved and / or simplified synthesis, a synthetic process with fewer degradation products, or a combination thereof, in combination with any of the preceding advantages or as independent advantages.

[0013] The RNAi agents herein include a sense strand and an antisense strand, each of which is an oligonucleotide. In some embodiments, the RNAi agents described herein also include a delivery moiety. As used herein, "nucleotide" refers to an organic compound having a nucleoside (e.g., a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar such as ribose or 2'-deoxyribose) and a phosphate group. A "nucleotide" can function as a monomer unit of a nucleic acid polymer, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0014] As used herein, "oligonucleotide" refers to a short nucleic acid compound (e.g., less than about 100 nucleotides in length). An oligonucleotide may be single-stranded (ss) or double-stranded (ds). An oligonucleotide may or may not have a duplex region. As a non-limiting set of examples, an oligonucleotide may 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).

[0015] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at the 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions of the nucleobase, sugar, or phosphate group.

[0016] As used herein, "modified internucleotide linkage" means an internucleotide linkage that has one or more chemical modifications when compared to a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage may be a non-naturally occurring linkage.

[0017] As used herein, a "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide may be a non-naturally occurring nucleotide. A modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, a modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0018] The term "percent sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleic acid bases in a candidate sequence that are identical to the nucleotides, nucleosides, or nucleic acid bases in the reference nucleic acid sequence, after optimally aligning the sequences and, if necessary, introducing gaps or overhangs to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software programs, such as those described in 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 W2.0, Clustal X2.0, or Megalign (DNASTAR) software. In one embodiment herein, sequence identity is calculated using Clustal W2.0 or Clustal X2.0. In another embodiment, sequence identity is calculated using Clustal W2.0. In another embodiment, sequence identity is calculated using Clustal X2.0. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, although the fragment of nucleic acid sequence within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleotide, nucleoside, or nucleic acid base is present in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity.The output is the percent identity of the subject sequence to the query sequence. In some embodiments, the percent sequence identity is the percentage of identical nucleotide residues between the two strands using a PID3 calculation, which is the number of identical nucleotide residues divided by the total number of nucleotides in the shorter of the two sequences, multiplied by 100. See, for example, Raghava, G., Barton, G. J. Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006).

[0019] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate. The 5'-phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates, such as 5'-methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). An oligonucleotide can have a phosphate analog at the 4'-carbon position of the sugar of the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of an oxymethyl group is attached to the sugar moiety (e.g., at the 4'-carbon) or an analog thereof. See, e.g., WO 2018 / 045317. Other modifications of the 5' end of oligonucleotides have also been developed (see, e.g., WO 2011 / 133871, U.S. Pat. No. 8,927,513, and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0020] 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 to allow hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotides herein comprise a targeting sequence having a region complementary to an mRNA target sequence.

[0021] As used herein, a "duplex" in reference to a nucleic acid or oligonucleotide, such as a sense strand or antisense strand, refers to a structure formed through hydrogen bonding of complementary base pairing of two antiparallel sequences of nucleotides under conditions suitable to promote such structure. A duplex can form even when there is no perfect complementarity between the two strands or when abasic nucleotides are present.

[0022] RNA interference is a specialized cellular process that utilizes RISC to degrade RNA in a sequence-dependent manner. As used herein, "RNAi agent" refers to an agent that includes either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion thereof is used by Argonaute 2 (Ago2) endonuclease to cleave the target mRNA, or (b) a single-stranded oligonucleotide having a single-stranded antisense strand, where the antisense strand (or a portion thereof) is used by Ago2 endonuclease to cleave the target mRNA. In some embodiments, the RNAi agent described herein also includes a delivery moiety.

[0023] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or halt the progression of a disorder or disease symptom disclosed herein, but does not necessarily indicate the complete disappearance of all disorder or disease symptoms. Treatment includes the administration of an RNAi agent or pharmaceutical composition thereof to treat a disease or condition in a mammal, including a human.

[0024] "Effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. The effective amount of an RNAi agent may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the RNAi agent to induce a desired response in the individual. An effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the RNAi agent.

[0025] Provided herein is an RNAi agent for reducing FAS gene expression, wherein the RNAi agent is a compound of formula I conjugated to R. [ka] wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand, R is conjugated to connection point E of formula I optionally via a linker, the sense strand and the antisense strand form a duplex region, the antisense strand comprises a region of complementarity to the FAS mRNA target sequence of SEQ ID NO: 1, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages.

[0026] Provided herein is an RNAi agent for reducing FAS gene expression, wherein the RNAi agent is a compound of formula Ia conjugated to R [ka] wherein R comprises an antisense strand and a sense strand, and R is conjugated to Formula Ia optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, and the antisense strand comprises a region of complementarity to the FAS mRNA target sequence of SEQ ID NO: 1, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages.

[0027] Disclosed herein is an RNAi agent for reducing FAS gene expression, the RNAi agent comprising a dsRNA including a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, the antisense strand comprising a region of complementarity of at least 15 nucleotides to the sequence set forth in SEQ ID NO: 1, and the sense strand and / or the antisense strand each optionally comprising one or more modified nucleotides and / or modified internucleotide linkages. In a further embodiment, the antisense strand comprises at least 15 nucleotides of a sequence in Table 2. In a further embodiment, the antisense strand comprises at least 18 nucleotides of a sequence in Table 2. In a further embodiment, the RNAi agent reduces FAS gene expression in cells expressing FAS by about 50% or more compared to a control. In a further embodiment, the RNAi agent reduces FAS gene expression by reducing FAS mRNA transcript levels, FAS protein levels, or both.

[0028] In further embodiments, the antisense strand is 15-50 nucleotides in length and / or the sense strand is 15-50 nucleotides in length. In further embodiments, the sense and / or sense strands are independently 15-30 nucleotides in length. In further embodiments, the antisense strand is 18-23 nucleotides in length. In further embodiments, the sense strand is 18-21 nucleotides in length.

[0029] In a further embodiment, the RNAi agent comprises an antisense strand comprising at least 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-112. In a further embodiment, the antisense strand comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-112.

[0030] In other further embodiments, the antisense strand comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 224-334, 337, 338, 573, and 577.

[0031] In other further embodiments, the sense strand comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 113-223, 335, 336, 572, and 576.

[0032] In a further embodiment, the antisense strand of the RNAi agent is 23 nucleotides in length. In a 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 sequences shown in Table 3A.

[0033] The sense strand and antisense strand of the RNAi agent disclosed herein do not need to be completely complementary.Therefore, in the RNAi agent disclosed herein, the duplex region between the sense strand and the antisense strand comprises 0, 1, 2 or 3 mismatches between the sense strand and the antisense strand.In a further embodiment, the duplex region between the sense strand and the antisense strand comprises 0, 1, 2 or 3 mismatches between the sense strand and the antisense strand.

[0034] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 129 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 240; b. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 116 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 227; c. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 151 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 262; d. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 128 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 239; and e. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 155 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 266 is selected from the group consisting of:

[0035] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 129 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 240; b. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 116 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 227; c. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 151 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 262; d. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 128 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 239; and e. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 155 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 266. is selected from the group consisting of:

[0036] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence comprising SEQ ID NO: 129 and a second nucleic acid sequence comprising SEQ ID NO: 240; b. a first nucleic acid sequence comprising SEQ ID NO: 116 and a second nucleic acid sequence comprising SEQ ID NO: 227; c. a first nucleic acid sequence comprising SEQ ID NO: 151 and a second nucleic acid sequence comprising SEQ ID NO: 262; d. a first nucleic acid sequence comprising SEQ ID NO: 128 and a second nucleic acid sequence comprising SEQ ID NO: 239; and e. A first nucleic acid sequence comprising SEQ ID NO: 155 and a second nucleic acid sequence comprising SEQ ID NO: 266 is selected from the group consisting of:

[0037] 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 further embodiments of the RNAi agent disclosed herein, each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide. In further embodiments, each nucleotide is a 2'-fluoro-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0038] In further embodiments of the RNAi agents disclosed herein, the antisense strand is 23 nucleotides in length, and each nucleotide in the antisense strand is a modified nucleotide, and the 2' fluoro-modified nucleotide is: 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 exists in.

[0039] In a further embodiment, the nucleotide that is not a 2' fluoro-modified nucleotide is a 2'-O-methyl-modified nucleotide.

[0040] In further embodiments of the RNAi agent disclosed herein, the sense strand and the antisense strand each independently comprise one or more modified internucleotide linkages, and each modified internucleotide linkage is a phosphorothioate linkage. In further embodiments, the sense strand and the antisense strand each independently comprise four phosphorothioate linkages. In further embodiments, the two terminal nucleotides at each of the 5'-end and 3'-end of each of the sense strand and the antisense strand are phosphorothioate linkages.

[0041] In other embodiments, the 5' nucleotide of the antisense strand comprises a phosphate group or a phosphate analog. As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of the oligonucleotide in place of the 5'-phosphate. The 5' phosphate analog can comprise a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). An oligonucleotide can have a phosphate analog at the 4'-carbon position of the sugar of the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of an oxymethyl group is attached to the sugar moiety (e.g., at the 4'-carbon) or an analog thereof. See, e.g., WO 2018 / 045317. Other modifications of the 5' end of oligonucleotides have also been developed (see, e.g., WO 2011 / 133871, U.S. Pat. No. 8,927,513, and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0042] In further embodiments of the RNAi agents disclosed herein, the antisense strand is selected from the group consisting of SEQ ID NOs: 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 39 8, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 475 6, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 555 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 ...7, 57, 57, 58, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 56, 56, 57, 57, 58, 59, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,

[0043] In further embodiments of the RNAi agents disclosed herein, the antisense strand is selected from the group consisting of SEQ ID NOs: 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 700, 701, 702, 703, 704, 705, 706, 70 35, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 750, 751, 752, 753, 754, 755, 756, 757, 759, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 6 03, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 7 71, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 813, 815, 817, 819, or a sequence having at least 90% sequence identity thereto.

[0044] In further embodiments, the sense strand is selected from the group consisting of SEQ ID NOs: 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 564, 565, 568, 574, 578, 580, 582, 584, or a sequence having at least 90% sequence identity thereto.

[0045] In further embodiments, the sense strand is selected from the group consisting of SEQ ID NOs: 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 4, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 77 8, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 809, 810, 811, 812, 814, 816, 818, or a sequence having at least 90% sequence identity thereto.

[0046] In further embodiments of the RNAi agents disclosed herein, the sense and antisense strands are a pair of oligonucleotide sequences selected from Table 4A or Table 4B, or sequences at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to a sequence in Table 4A or Table 4B. In further embodiments, one, two, or three mismatches are introduced into the sense strand of a pair in Table 4A, Table 4B, or Table 7. In further embodiments, one, two, or both terminal nucleotides at the 5' end of the antisense strand are altered.

[0047] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 339 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 340; b. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 341 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 342; c. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 343 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 344; d. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 345 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 346; e. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 347 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 348; f. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 349 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 350; g. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 353 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 354; h. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 363 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 364; and i. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 381 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 382. is selected from the group consisting of:

[0048] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 564 or 809 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 571; b. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 568 or 811 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 567; c. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 580 or 814 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 581 or 815; d. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 582 or 816 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 583 or 817; and e. a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 584 or 818 and a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 585 or 819. is selected from the group consisting of:

[0049] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 564 or 809 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 571; b. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 568 or 811 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 567; c. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 580 or 814 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 581 or 815; d. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 582 or 816 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 583 or 817; and e. a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 584 or 818 and a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 585 or 819. is selected from the group consisting of:

[0050] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence comprising SEQ ID NO: 564 or 809 and a second nucleic acid sequence comprising SEQ ID NO: 571; b. a first nucleic acid sequence comprising SEQ ID NO: 568 or 811 and a second nucleic acid sequence comprising SEQ ID NO: 567; c. a first nucleic acid sequence comprising SEQ ID NO: 580 or 814 and a second nucleic acid sequence comprising SEQ ID NO: 581 or 815; d. a first nucleic acid sequence comprising SEQ ID NO: 582 or 816 and a second nucleic acid sequence comprising SEQ ID NO: 583 or 817; and e. A first nucleic acid sequence comprising SEQ ID NO: 584 or 818 and a second nucleic acid sequence comprising SEQ ID NO: 585 or 819. is selected from the group consisting of:

[0051] In some embodiments, the RNAi agent comprises a sense strand comprising a first nucleic acid sequence and an antisense strand comprising a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are a. a first nucleic acid sequence consisting of SEQ ID NO: 564 or 809 and a second nucleic acid sequence consisting of SEQ ID NO: 571; b. a first nucleic acid sequence consisting of SEQ ID NO: 568 or 811 and a second nucleic acid sequence consisting of SEQ ID NO: 567; c. a first nucleic acid sequence consisting of SEQ ID NO: 580 or 814 and a second nucleic acid sequence consisting of SEQ ID NO: 581 or 815; d. a first nucleic acid sequence consisting of SEQ ID NO: 582 or 816 and a second nucleic acid sequence consisting of SEQ ID NO: 583 or 817; and e. a first nucleic acid sequence consisting of SEQ ID NO: 584 or 818 and a second nucleic acid sequence consisting of SEQ ID NO: 585 or 819 is selected from the group consisting of:

[0052] In further embodiments, the 5' terminal nucleotide of the antisense strand is substituted such that the final sequence contains a vinyl phosphonate, a phosphate group, or an OH group, e.g., SEQ ID NOs: 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404 , 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474 , 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544 For the antisense sequences of 4, 546, 548, 550, 552, 554, 556, 558, 560, 562, 563, 566, 567, 569, 570, 571, 575, 579, 581, 583, 585, or sequences having at least 90% sequence identity thereto, the 5' phosphate group is replaced with an OH group.

[0053] In other embodiments, the RNAi agents disclosed herein comprise a compound of formula I conjugated to R. [ka] wherein R is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides having complementarity to the FAS mRNA target sequence of SEQ ID NO: 1, wherein the sense strand and antisense strand form a region of complementarity of at least 15 nucleotides, wherein the sense strand and antisense strand are each independently 18-23 nucleotides in length, optionally, the sense strand and antisense strand each independently comprise one or more modified nucleotides, and optionally, the sense strand and antisense strand each independently comprise one or more modified internucleotide linkages, and R is optionally conjugated to Formula I via a linker. In further embodiments, the sense strand or antisense strand is selected from Table 2, Table 3A, Table 3B, Table 4A, Table 4B, Table 7, or Table 8 disclosed herein. In other embodiments, the antisense or antisense strand of the RNAi agent has a sequence with 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 2, Table 3A, Table 3B, Table 4A, Table 4B, Table 7, or Table 8 herein.

[0054] In other embodiments, the RNAi agents disclosed herein comprise a linker. In further embodiments, R is conjugated to Formula I via a linker. In other further embodiments, R is conjugated to Formula I via a linker. In further embodiments, the linker is a linker of Formula II having connection points A and B. [ka] or the linker comprises Formula III having attachment points C and D; a. Formula I is conjugated to Formula II at attachment point A, and Formula II is conjugated to a phosphate group at attachment point B, and the phosphate group is conjugated to R; or b. Formula I is conjugated to Formula III at connection point C, and Formula III is conjugated to a phosphate group at connection point D, which is further conjugated to R.

[0055] In other embodiments where the RNAi agent comprises a linker, R is conjugated to Formula I via a linker, and the linker is Formula III having attachment points C and D. [ka] wherein Formula I is conjugated to Formula III at connection point C, Formula III is conjugated to a phosphate group at connection point D, which is further conjugated to R.

[0056] The sense and antisense strands of FAS RNAi agents can be synthesized using any nucleic acid polymerization method known in the art, such as solid-phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercially available synthesizers, such as the MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems, can be used. Phosphorothioate linkages can be introduced using sulfurizing reagents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione). It is well known to synthesize modified oligonucleotides using similar techniques as well as commercially available modified amidite and controlled-pore glass (CPG) products.

[0057] In yet another embodiment, the RNAi agent can reduce the expression of the FAS gene in liver cells. In another embodiment, the RNAi agent disclosed herein is for use in therapy. In a further embodiment, the use is for the treatment of AIH.

[0058] RNAi agent can be formulated into pharmaceutical composition.Therefore, the present specification discloses pharmaceutical composition comprising the RNAi agent disclosed herein and one or more pharmaceutically acceptable excipients.Pharmaceutical composition can be prepared by the method well known in the art (for example, Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A.Loyd et al., Academic Press).

[0059] In another embodiment, there is provided a use of an RNAi agent herein for the manufacture of a medicament for the treatment of AIH.

[0060] In another embodiment, there is provided a method of treating AIH in a patient in need thereof, comprising administering a FAS RNAi agent or pharmaceutical composition thereof disclosed herein.

[0061] The RNAi agent may be administered to the patient intravenously or subcutaneously.

[0062] RNAi dosing regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0063] Dosage values ​​can vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0064] In another embodiment, a method of reducing FAS expression in a cell is provided, comprising contacting the cell with an RNAi agent disclosed herein and incubating the cell for a time sufficient to reduce the level of FAS mRNA by at least 50% compared to untreated or control-treated cells. [Example]

[0065] Certain abbreviations are defined as follows: "1,2-DCE" refers to 1,2-dichloroethane, "DCM" refers to dichloromethane, "N,N-diisopropylethylamine (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, and "EtOAc" refers to ethyl acetate. 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, and "HBTU" refers to O-(benzotriazol-1-yl)-N,N,N',"N'-tetramethyluronium hexafluorophosphate," "HOBt" refers to 1-hydroxybenzotriazole hydrate, "HPRT" refers to hypoxanthine-guanine phosphoribosyltransferase, "IPA" refers to isopropanol and isopropyl alcohol, "LDHA" refers to lactate dehydrogenase-A, "MeCN" refers to acetonitrile, "MeOH" refers to methanol and methyl alcohol, "MWCO" refers to molecular weight cut-off, "NHS" refers to N-hydroxysuccinimide, "optical density (OD)" refers to optical density, "PBS" refers to phosphate-buffered saline, "PhSiH3" refers to phenylsilane, "PTS" refers to portable endotoxin test system, and "siRNA" refers to small interfering ribonucleic acid. "TFA" refers to trifluoroacetic acid, "THF" refers to tetrahydrofuran, "TLC" refers to thin-line chromatography, and "TMP" refers to 2,2,6,6-tetramethylpiperidine.

[0066] Delivery moieties comprising Formula I can be made by the following non-limiting synthetic steps and schemes.

[0067] [ka]

[0068] Step A in Scheme 1 depicts the cyclization of compound (1) using trimethyl trifluoromethanesulfonate in a solvent such as 1,2-DCE to give compound (2). Step B depicts the addition of hex-5-en-1-ol to compound (2) using trimethylsilyl trifluoromethanesulfonate in a solvent such as 1,2-DCE to give compound (3). Step C depicts the oxidation of compound (3) using a suitable oxidizing agent such as sodium periodate with a catalyst such as ruthenium(III) chloride to give compound (4).

[0069] [ka]

[0070] Step A in Scheme 2 illustrates the amide coupling of compound (5) with tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate using HBTU and HOBt in a solvent such as DMF with a suitable base such as DIEA to obtain compound (6). Step B illustrates the basic hydrolysis of compound (6) using a base such as aqueous NaOH in a THF and MeOH solvent system to obtain compound (7). Step C illustrates the amide coupling of compound (7) with allyl 11-aminoundecanoate hydrochloride using HATU in a solvent such as DMF with a suitable base such as DIEA to obtain compound (8). Step D illustrates the acidic deprotection of compound (8) with TFA in a solvent such as DCM to obtain compound (9). Step E illustrates the amide coupling of compound (9) with compound (4) using EDC and HOBt in a solvent such as DCM to obtain compound (10). Step F illustrates the deprotection of compound (10) with tetrakis(triphenylphosphine)palladium and PhSiH in a solvent such as DCM to give compound (11). Step F illustrates the coupling of compound (11) with NHS using EDC in a solvent such as DCM to give compound (12).

[0071] [ka]

[0072] Steps A-C of Scheme 3 are essentially similar to steps C-E of Scheme 2, starting from compound 7 to give compounds 13, 14, and 15. Step D shows the hydrogenation of compound 15 using palladium on carbon in a solvent such as MeOH to give compound 16. Step E is essentially similar to the preparation of step G of Scheme 2 to give compound 17.

[0073] [ka]

[0074] Steps AI of Scheme 4 consist of a series of amide couplings and deprotections using methods essentially similar to those shown in Schemes 2 and 3, starting from compound (18) to give compound (27).

[0075] [ka]

[0076] Steps A through C of Scheme 5 show essentially similar methods as those shown in Steps G through I of Scheme 4, starting from compound (24) to give compound (30).

[0077] [ka]

[0078] Step A of Scheme 6 depicts the protection of compound 31 using DMTCl with a suitable base such as DIEA in a solvent such as DCM to give compound 32. Step B depicts the amide coupling of compound 32 with piperidin-4-ylmethanol using HBTU and HOBt with TMP in a solvent such as DCM to give compound 33. Step C depicts the deprotection of compound 33 with 20% piperidine in DMF to give compound 34.

[0079] [ka]

[0080] Step A of Scheme 7 is essentially similar to Step A of Scheme 2, and involves the coupling of compound 16 with compound 34 to give compound 35. Step B illustrates the formation of compound 36 by adding succinic anhydride to compound 35 with a base system of TEA and DMAP in a suitable solvent such as DCM. Step C illustrates the loading of compound 36 onto the 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 give compound 37.

[0081] Preparation 1 Methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate [ka]

[0082] 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) is added trimethylsilyl trifluoromethanesulfonate (6.5 mL, 35 mmol). The mixture is heated to 50 °C and stirred for 18 h. After this time, the mixture is diluted with DCM (200 mL), washed with saturated aqueous NaHCO (200 mL) and saturated aqueous sodium chloride (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM to give the title compound (6.434 g, 84%). ES / MS m / z 330 (M+H).

[0083] Preparation 2 (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate methyl [ka]

[0084] 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) is added hex-5-en-1-ol (22.2 mL, 185 mmol), followed by activated powdered 4 Å molecular sieves (15.6 g). The suspension is stirred at ambient temperature for 30 min, then trimethylsilyl trifluoromethanesulfonate (19 mL, 101.9 mmol) is added. The mixture is stirred at ambient temperature for 18 h. After this time, the solution is filtered through diatomaceous earth and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 30-100% EtOAc / hexane to give the title compound (34.76 g, 86%). ES / MS m / z 430.4 (M+H).

[0085] Preparation 3 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid [ka]

[0086] A solution of methyl (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate (34.76 g, 80.93 mmol) in MeCN (174 mL) and DCM (174 mL) is cooled to 0 °C. A solution of sodium periodate (22.4 g, 104.7 mmol) is added, and stirring is continued at 0 °C for 10 minutes. After this time, ruthenium(III) chloride (270 mg, 1.3 mmol) is added, and the mixture is stirred while warming to ambient temperature. After stirring for 2 hours, additional sodium periodate (66 g, 308.4 mmol) is added, and stirring is continued for 18 hours. After this time, the mixture is extracted with 3:1 CHCl:IPA (2 × 500 mL), washed with saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-40% MeOH / DCM to give the title compound (29.75 g, 82%). ES / MS m / z 448.4 (M+H).

[0087] Preparation 4 Benzyl 6-aminohexanoate hydrochloride [ka]

[0088] To a suspension of 6-aminohexanoic acid (5.00 g, 38.1 mmol) in THF (38 mL) is added benzyl alcohol (47 mL, 453.7 mmol) and the mixture is cooled to 0 °C. Thionyl chloride (8.6 mL, 120 mmol) is added dropwise and the mixture is stirred for 18 hours while warming to ambient temperature. After this time, ether (166 mL) is added and the reaction vessel is transferred to a -20 °C freezer for 1 hour. After this time, the solid precipitate is collected by filtration to give the title compound (8.57 g, 81%). ES / MS m / z 222 (M+H).

[0089] Preparation 5 Benzyl 11-aminoundecanoate hydrochloride [ka]

[0090] The title compound is prepared from 11-aminoundecanoic acid in a manner essentially similar to the method of Preparation 4. ES / MS m / z 292.2 (M+H).

[0091] Preparation 6 Allyl 11-aminoundecanoate hydrochloride [ka]

[0092] 11-Aminoundecanoic acid (9.00 g, 44.7 mmol) in allyl alcohol (42 mL) is placed in a vessel and the mixture is cooled to 0 °C. Thionyl chloride (6.5 mL, 89.4 mmol) is added, and the mixture is stirred for 18 hours while warming to ambient temperature. After this time, the mixture is concentrated in vacuo, and ether (200 mL) is added to the residue to give a white suspension. The mixture is stirred at ambient temperature for 10 minutes, and the solid precipitate is collected by filtration to give the product (12.0 g, 97%). ES / MS m / z 242.2 (M+H).

[0093] Preparation 7 (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9ylmethoxycarbonylamino)propanoic acid [ka]

[0094] To a stirred solution of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-propanoic acid (40 g, 0.122 mol) in dry DCM (400 mL) is added DIEA (64 mL, 0.366 mol) at 0° C. under an inert atmosphere. To this is slowly added a solution of DMTCl (49.6 g, 0.146 mol) in DCM (200 mL). The resulting reaction mixture is warmed to ambient temperature and stirred for 16 hours. After this time, the reaction mixture is diluted with water (12.5 vol) and extracted with DCM (25 vol). The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting crude product is washed with 10% EtOAc / hexane (12.5 vol) and dried under vacuum to afford the title compound as a light brown solid (62 g, crude). This material was used in the next step without further purification. TLC: 5% MeOH / CH2Cl2 (Rf: 0.5) UV, 254nM.

[0095] Preparation 8 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]carbamate [ka]

[0096] To a stirred solution of (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9ylmethoxycarbonylamino)propanoic acid (62 g, 0.103 mol) in DCM (750 mL) is 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) under an inert atmosphere at 0° C. The resulting reaction mixture is allowed to warm to ambient temperature and stirred for 4 hours. After this time, the reaction mixture is diluted with water (8 vol) and extracted with DCM (15 vol). The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 20-40% EtOAc / hexanes and 1% MeOH / DCM to give the title compound (40 g, 52% over two steps). 1H 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.

[0097] Preparation 9 (2S)-2-Amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1-piperidyl]propan-1-one [ka]

[0098] A solution of 20% piperidine in DMF (400 mL) is slowly added to 9H-fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]carbamate (40 g, 0.055 mol) under an inert atmosphere at 0 °C. The resulting reaction mixture is stirred at ambient temperature for 1 h. After this time, the mixture is diluted with water (15 vol) and extracted with EtOAc (30 vol). The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 1-8% MeOH / DCM to afford the title compound as an off-white solid (13 g, 47%). ES / MS m / z 1009.5 (2M+H).

[0099] Preparation 10 Methyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate [ka]

[0100] 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) is 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) is added and the mixture is stirred at ambient temperature for 5 minutes. After this time, tert-butyl N-[2-[2-(ter t-Butoxycarbonylamino)ethylamino]ethyl]carbamate (8.94 g, 29.5 mmol) is added in one portion and stirring is continued at ambient temperature. After stirring for 18 h, the mixture is diluted with EtOAc (400 mL), washed with water (2 × 400 mL) and saturated aqueous sodium chloride solution (400 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 40–100% EtOAc / hexane to give the title compound (13.01 g, 89%). ES / MS m / z 547.40 (M+H).

[0101] Preparation 11 (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid [ka]

[0102] A flask is charged with methyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate (13.01 g, 23.8 mmol), THF (120 mL), and MeOH (120 mL). 1N NaOH (71 mL, 71 mmol) is added, and the mixture is stirred at ambient temperature. After 1 hour, the mixture is concentrated in vacuo and redissolved in water (300 mL). 5N HCl (12 mL) is added to bring the pH to 4. The mixture is extracted with DCM (3 x 300 mL), and the combined organic layers are washed with saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated to give the title compound (12.41 g, 98%). ES / MS m / z 531.60 (MH).

[0103] Preparation 12 Allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoate [ka]

[0104] A flask containing (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (500 mg, 0.94 mmol) and allyl 11-aminoundecanoate hydrochloride (313 mg, 1.13 mmol) was charged with DMF (6.25 mL) and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (428 mL). g, 1.12 mmol) is added. After adding DIEA (0.5 mL, 3 mmol), the mixture is stirred at ambient temperature for 18 hours. After this time, the mixture is diluted with EtOAc (200 mL), washed with water (3 x 200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 40-100% EtOAc / hexane to give 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).

[0105] Preparation 13 Allyl (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)undecanoate [ka]

[0106] 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 DCM (15 mL) is added TFA (15 mL). The mixture is stirred at room temperature. After 1.5 h, the mixture is concentrated in vacuo. The residue is dissolved in MeOH and loaded onto an ion exchange cartridge. The cartridge is eluted with MeOH (150 mL) followed by 7N NH3 / MeOH (150 mL). The basic fractions are concentrated in vacuo to give the title compound (410 mg, 99%). ES / MS m / z 456.4 (M+H).

[0107] Preparation 14 Allyl 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]undecanoate [ka]

[0108] A flask is 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-oxopentanamido)undecanoate (150 mg, 0.33 mmol). DCM (3.35 mL) is 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 is stirred at ambient temperature for 18 h. After this time, the solution is 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 is dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM to give the title compound (424 mg, 74%). ES / MS m / z 872.80 (M+2H) / 2.

[0109] Preparation 15 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]undecanoic acid [ka]

[0110] To a solution of allyl 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate (354 mg, 0.20 mmol) in 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 ambient temperature for 2 h and then diluted with saturated aqueous NaHCO3 (100 mL). 1 N NaOH (15 mL) was added to bring the pH to approximately 10. The aqueous solution is washed with DCM (3 x 100 mL) and then acidified with concentrated HCl (5 mL) and then 5N aqueous HCl (15 mL). The aqueous layer is extracted with DCM (100 mL), and the organic layer is dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-20% MeOH / DCM to give the title compound (151 mg, 44%). ES / MS m / z 852.60 (M+2H) / 2.

[0111] Preparation 16 (2,5-Dioxopyrrolidin-1-yl)11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate [ka]

[0112] To a reaction vial, 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]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) were added. DCM (0.3 mL) was added, and the mixture was stirred at ambient temperature. After 18 h, the mixture is loaded directly onto a silica gel cartridge and the crude mixture is purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM to give the title compound (49 mg, 93%). ES / MS m / z 901.40 (M+2H) / 2.

[0113] Preparation 17 Benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoate [ka]

[0114] The title compound is 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 similar to that of Preparation 10. ES / MS m / z 736.40 (M+H).

[0115] Preparation 18 Benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoate tris(trifluoroacetic acid) [ka]

[0116] 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) is added TFA (16 mL, 210.2 mmol). The mixture is stirred at ambient temperature for 24 h. After this time, additional TFA (16 mL, 210.2 mmol) is added and stirring is continued for another 2 h. After this time, the mixture is concentrated in vacuo. The resulting residue is azeotroped with toluene (2 × 30 mL). The resulting oil is further dried in a vacuum oven at 40 °C for 4 h to give the title compound (28.08 g, 58% purity considering residual toluene, 99+%). ES / MS m / z 436.40 (M+H). The compound is dissolved in 70 mL of DMF to make a 0.3 M solution which is used in the next step.

[0117] Preparation 19 Benzyl 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate [ka]

[0118] The title compound is prepared from 5-[3-acetamido-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 tristrifluoroacetate in a manner essentially similar to that of Preparation 10. ES / MS m / z 862 (M+2H) / 2.

[0119] Preparation 20 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]hexanoic acid [ka]

[0120] Palladium on carbon (1.90 g, 0.89 mmol, 5% by weight, 50% wet) was placed in a round-bottom flask, and the vessel was evacuated and refilled with nitrogen three times. A solution of benzyl 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate (15.41 g, 8.94 mmol) in MeOH (178 mL) was added via syringe. The flask was evacuated and refilled with 1 atm of hydrogen, and the mixture was stirred at ambient temperature under 1 atm of hydrogen for 18 h. After this time, the mixture is filtered through diatomaceous earth and the filtrate is concentrated in vacuo to give the title compound (13.85 g, 95%). ES / MS m / z 817.2 (M+2H) / 2.

[0121] Preparation 21 (2,5-Dioxopyrrolidin-1-yl)6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate [ka]

[0122] The title compound is prepared from 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid in a manner essentially similar to that of Preparation 16. ES / MS m / z 866.20 (M+2H) / 2.

[0123] Preparation 22 Benzyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate [ka]

[0124] The title compound is 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 similar to that of Preparation 12. ES / MS m / z 623.6 (M+H).

[0125] Preparation 23 Benzyl (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxopentanoate tris(trifluoroacetate) salt [ka]

[0126] The title compound is prepared from benzyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate in a manner essentially similar to the method of Preparation 18. ES / MS m / z 323.2 (M+H).

[0127] Preparation 24 Benzyl (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoate [ka]

[0128] The title compound is prepared from 5-(tert-butoxycarbonylamino)pentanoic acid and benzyl (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoate tris(trifluoroacetate) salt in a manner essentially similar to that of Preparation 10. ES / MS m / z 920.6 (M+H).

[0129] Preparation 25 Benzyl (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoate tris(trifluoroacetic acid) salt [ka]

[0130] The title compound is prepared from benzyl (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoate in a manner essentially similar to the method of Preparation 18. ES / MS m / z 620.4 (M+H).

[0131] Preparation 26 Benzyl (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-pentanoate [ka]

[0132] The title compound is prepared from 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid and benzyl (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoate tris(trifluoroacetate) salt in a manner essentially similar to that of Preparation 10. ES / MS m / z 954.80 (M+2H) / 2.

[0133] Preparation 27 (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-oxopentanoic acid [ka]

[0134] A round-bottom flask was charged with palladium on carbon (467 mg, 0.22 mmol, 5% by weight, 50% wet). The flask was evacuated and refilled with nitrogen three times. A solution of benzyl (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-pentanoate (4.19 g, 2.20 mmol) in MeOH (44 mL) was added via syringe, followed by the addition of three drops of acetic acid. The flask was evacuated and refilled with 1 atm of hydrogen, and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After 2 hours, the mixture is filtered through diatomaceous earth and the filtrate is concentrated in vacuo to give the title compound (3.99 g, 99+%). ES / MS m / z 909.6 (M+2H) / 2.

[0135] Preparation 28 Benzyl 6-[[(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]hexanoate [ka]

[0136] The title compound is prepared from (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-pentanoic acid and benzyl 6-aminohexanoate hydrochloride in a manner essentially similar to the method of Preparation 10. ES / MS m / z 1011.6 (M+2H) / 2.

[0137] Preparation 29 6-[[(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-oxopentanoyl]amino]hexanoic acid [ka]

[0138] A round-bottom flask was charged with palladium on carbon (24 mg, 0.01 mmol, 5% by weight, 50% wet), the flask was evacuated, and backfilled with nitrogen. A solution of benzyl 6-[[(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]hexanoate (222 mg, 0.11 mmol) in MeOH (2.2 mL) was added via syringe, followed by the addition of 3 drops of acetic acid. The flask is evacuated and backfilled with 1 atm of hydrogen, and the mixture is stirred at ambient temperature under 1 atm of hydrogen. After 5 h, the flask is purged with nitrogen, and the mixture is filtered through diatomaceous earth. The filtrate is concentrated in vacuo to give the title compound (180 mg, 85%). ES / MS m / z 966.2 (M+2H) / 2.

[0139] Preparation 30 (2,5-Dioxopyrrolidin-1-yl)6-[[(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]hexanoate [ka]

[0140] The title compound is prepared from 6-[[(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]hexanoic acid in a manner essentially similar to that of Preparation 16. ES / MS m / z 1014.6 (M+2H) / 2.

[0141] Preparation 31 Benzyl 11-[[(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]undecanoate [ka]

[0142] The title compound is prepared from (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-pentanoic acid and benzyl 11-aminodecanoate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1046.6 (M+2H) / 2.

[0143] Preparation 32 11-[[(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-oxopentanoyl]amino]undecanoic acid [ka]

[0144] Palladium on carbon (35 mg, 0.02 mmol, 5% by weight, 50% wet) was added to a round-bottom flask, which was then evacuated and refilled with nitrogen three times. A solution of benzyl 11-[[(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]undecanoate (285 mg, 80% pure, 0.11 mmol) was added via syringe. The vessel was evacuated and refilled with 1 atm of hydrogen, and the mixture was then stirred at ambient temperature under 1 atm of hydrogen. After stirring for 3 hours, the flask is purged with nitrogen and the mixture is filtered through diatomaceous earth. The filtrate is concentrated to give the title compound (213 mg, 79% purity, 77%). ES / MS m / z 1001.20 (M+2H) / 2.

[0145] Preparation 33 (2,5-Dioxopyrrolidin-1-yl)11-[[(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]undecanoate [ka]

[0146] The title compound is prepared from 11-[[(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]undecanoic acid in a manner essentially similar to the method of Preparation 16. ES / MS m / z 1050(M+2H) / 2

[0147] Preparation 34 [5-acetamido-6-[5-[2-[[(4S)-4-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxo-pentoxy]-3,4-diacetoxytetrahydropyran-2-yl]acetate methyl ester [ka]

[0148] The title compound is prepared from 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-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-piperidyl]propan-1-one in a manner essentially similar to that of Preparation 10. ES / MS m / z 1059.2 (M-2H) / 2.

[0149] Preparation 35 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propanoyl]-4-piperidyl]methoxy]-4-oxo-butanoic acid [ka]

[0150] [5-acetamido-6-[5-[2-[[(4S)-4-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl in DCM (11 mL) To a solution of methyl 2-[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxo-pentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]acetate (1.194 g, 0.56 mmol), succinic anhydride (113 mg, 1.13 mmol), TEA (0.4 mL, 3 mmol), and DMAP (213 mg, 1.69 mmol) are added. The mixture is stirred at ambient temperature for 1 hour. After this time, the mixture is diluted with saturated NH4Cl (200 mL) and extracted with DCM (3 x 200 mL) and 3:1 CHCl3:IPA (200 mL). The organic layers are combined, dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-40% MeOH / DCM, and the resulting product is dried in a vacuum oven at 40 °C for 3 h to give the title compound (1.081 g, 86%). ES / MS m / z 1109.60 (M-2H) / 2.

[0151] Preparation 36 Resin Loading [ka]

[0152] A solution of 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propanoyl]-4-piperidyl]methoxy]-4-oxo-butyric acid (1.00 g, 0.61 mmol) in MeCN (6 mL) and DCM (1 mL) is transferred to a 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) are added to the vessel, and the cartridge is shaken at ambient temperature for 5 minutes. After this, 1000 Å LCAA controlled pore glass resin (5.39 g, 90 μmol / g loading, purchased from ChemGenes) is added, and the mixture is shaken at ambient temperature for 18 hours. After this, the cartridge is drained by suction, and the resin is washed with DCM (10 mL) by shaking for 10 minutes. The cartridge is drained, and the washing and draining procedure is repeated with 10% MeOH / DCM (10 mL) and EtO (10 mL). After draining, a solution of acetic anhydride (6.4 mL), pyridine (20 mL), and TEA (0.22 mL) is added, and the cartridge is shaken for 2 hours. After this, the cartridge is drained, and the above washing and draining procedure is repeated using DCM (10 mL), 10% MeOH / DCM (10 mL), and diethyl ether (10 mL). After draining, the resin is dried under vacuum for 30 minutes. The resin loading is measured using a standard trityl assay. The resin loading was calculated to be 34.7 μmol / g.

[0153] Preparation 37 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-oxopentanoyl]amino]ethoxy]acetate [ka]

[0154] The title compound is prepared from (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-pentanoic acid and benzyl 2-(2-aminoethoxy)acetate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1005.2 (M+2H / 2).

[0155] Preparation 38 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-oxopentanoyl]amino]ethoxy]acetic acid [ka]

[0156] 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]benzyl acetate (0.120 mmol, 240 mg) is mixed with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 ml). The mixture is hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 48 minutes, filtered through diatomaceous earth, and concentrated in vacuo to give the title compound as a gray solid (187 mg, 82%). ES / MS m / z 960.0 (M+2H / 2).

[0157] Preparation 39 (2,3,5,6-Tetrafluorophenyl)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-oxopentanoyl]amino]ethoxy]acetic acid [ka]

[0158] 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 dropwise (2,3,5,6-tetrafluorophenyl)-2,2,2-trifluoroacetic acid (0.383 mmol, 100 mg). The mixture was stirred at ambient temperature for 16 hours. The reaction mixture is directly purified by silica gel flash chromatography eluting with 0% to 50% MeOH / DCM to give the title compound as a tan solid (197 mg, 99%). ES / MS m / z 1034.0 (M+2H / 2).

[0159] Preparation 40 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-oxopentanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]acetate [ka]

[0160] The title compound is prepared from (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-pentanoic acid and benzyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1049.0 (M+2H / 2).

[0161] Preparation 41 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-oxopentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid [ka]

[0162] 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]benzyl acetate (0.118 mmol, 247 mg) is mixed with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 mL). The mixture is hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 1 hour, filtered through diatomaceous earth, and concentrated in vacuo to give the title compound as a gray solid (227 mg, 96%). ES / MS m / z 1004.0 (M+2H / 2).

[0163] Preparation 42 (2,3,5,6-Tetrafluorophenyl)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-oxopentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid [ka]

[0164] 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 dropwise (2,3,5,6-tetrafluorophenyl)-2,2,2-trifluoroacetic acid (0.443 mmol, 116 mg). The mixture is stirred at ambient temperature for 16 hours. The reaction mixture is directly purified by silica gel flash chromatography eluting with 0% to 50% MeOH / DCM to give the title compound as a tan solid (174 mg, 73%). ES / MS m / z 1078.2 (M+2H / 2).

[0165] Example 1: Conjugation Protocol For the synthesis of the GalNAc-conjugated sense strand, the sense strand bearing 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 GalNAc ligand (5 equivalents). The mixture was shaken at ambient temperature for 16 hours. After this time, the mixture was transferred to a 15 mL Falcon tube, ammonium hydroxide (28% by weight) was added, and the mixture was shaken at ambient temperature for 2 hours. The ammonia was then removed in vacuo. The residue was purified by ion exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15% MeCN / 20 mM NaH2PO4, 1 M NaBr; 35-55% B over 5 CV at 8 mL / min, column temperature 60 °C. Desired fractions were pooled and desalted by Eppendorf centrifugation or spin filtration using a desalting column. After desalting, the material was collected and the OD and volume were measured to obtain the concentration.

[0166] Alternatively, conjugation was performed to the 5' position of the sense strand by immobilizing the GalNAc ligand on microporous polystyrene resin or controlled pore glass and synthesizing it using established solid-phase oligonucleotide synthesis methods with 5'-CE(β-cyanoethyl) phosphoramidite.

[0167] Alternatively, the GalNAc ligand was converted to a suitable phosphoramidite and delivered to the 5' position of the sense strand using standard phosphoramidite chemistry.

[0168] Example 2: Annealing To generate sense and antisense siRNA duplexes, the following procedure was performed: To the Falcon tube containing the oligonucleotide sense-GalNAc conjugate, the corresponding antisense oligonucleotide (1 equivalent) was added, vortexed for 10 seconds, and then spin-filtered through a 100K MWCO Amicon filter unit to remove particulates. The filtrate was collected and concentrated in vacuo in a Genevac evaporator. The residue was reconstituted in 1x PBS, filtered through a 0.2μ filter, and the OD and volume were measured to obtain the concentration.

[0169] Endotoxin testing was performed using Limulus amebocyte lysate on an Endosafe®-nexgen PTS instrument.

[0170] [Table 1-1]

[0171] [Table 1-2]

[0172] Example 3: General Procedure for Oligonucleotide Synthesis Using GalNAc-Functionalized CPG Oligos were synthesized using phosphoramidite chemistry on a MerMade® 12 instrument. The sense strand was synthesized from a pre-functionalized GalNAc solid support, and the antisense strand was synthesized using a standard support pre-loaded with the first nucleotide of the oligo sequence. The oligos were cleaved and deprotected using concentrated ammonium hydroxide solution (28% by weight) and purified by ion exchange chromatography using the conditions described above. Desalting, annealing, and endotoxin testing were performed.

[0173] Antisense oligonucleotide sequences were designed using 15-50 nucleotides of the following FAS transcript (SEQ ID NO: 1), in which T nucleotides were replaced with U nucleotides, and one or more nucleotides and one or more internucleotide linkages were further modified, as needed, as described herein.

[0174] Homo sapiens FAS death receptor (FAS) transcript SEQ ID NO: 1 [ka] [ka]

[0175] Exemplary antisense strand sequences of 18 nucleotides in length are shown below in Table 2, which may optionally be further modified and synthesized and incorporated into RNAi agents as described herein.

[0176] [Table 2-1]

[0177] [Table 2-2]

[0178] [Table 2-3]

[0179] [Table 2-4]

[0180] [Table 3-1]

[0181] [Table 3-2]

[0182] [Table 3-3]

[0183] [Table 3-4]

[0184] [Table 3-5] GNA denotes glycol nucleic acid nucleotide (structure shown in Table 3B). (AP) refers to apurinic / apyrimidinic residues, also called abasic residues (structures shown in Table 3B).

[0185] [Table 4]

[0186] Example 4: In vitro knockdown of hFAS in HepG2 cells The RNAi agents in Tables 4A and 4B were tested in HepG2 cells. Reverse transfection was performed by adding 0.3 μl of Lipofectamine RNAiMAX along with 24.7 μl of Opti-MEM per well to 25 μl of each 4x human FAS-GalNAc siRNA in individual wells of a 96-well collagen I-coated plate. The mixture was incubated at room temperature for 20 minutes, and then 50 μl of growth medium containing HepG2 cells at 300,000 cells / mL was added to the human FAS-GalNAc siRNA / RNAiMAX mixture. The final siRNA concentration was 500 nM for single-concentration screening, as described above. After incubating the cells for 24–48 hours, RNA was isolated using the Quick-RNA 96 Kit. The RNA was then stored at -80°C or subjected to cDNA synthesis. Briefly, cDNA was synthesized from the purified RNA using Fast Advanced RT Master Mix (Invitrogen). A master mix of 5 μl of 2x Fast Advanced RT buffer and 0.5 μl of 20x Fast Advanced RT enzyme mix was prepared per reaction. 5.5 μl of master mix and 4.5 μl of RNA were mixed for a final volume of 10 μl. cDNA was generated using the ProFlex PCR System (Life Technologies) by the following steps: 37°C for 30 minutes, 95°C for 5 minutes, and a 4°C hold.

[0187] Two microliters of cDNA was added to a master mix containing 2.5 μl of HO, 0.5 μl of 20x TaqMan Gene Expression Assay Buffer (Life Technologies), and 5 μl of 2x TaqMan Universal PCR Master Mix (Life Technologies). The following PCR cycles were completed using a QuantStudio 7 Flex Real-Time PCR System (Life Technologies): 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. TaqMan gene expression assays were performed. Data analysis was performed using the ddCt method.

[0188] For concentration-response curves, selected siRNAs from each assay were used to determine IC50s using 1:3 serial dilutions to final concentrations of FAS-GalNAc RNAi agents of 200 nM, 67 nM, 22 nM, 7.41 nM, 2.47 nM, 0.82 nM, and 0.27 nM. IC50 values ​​were calculated using a four-parameter fit model using XLFit.

[0189] The data are shown in Table 5.

[0190] Example 5: In vivo knockdown in hFAS-AAV-treated mice using the FAS RNAi agent described herein After anesthesia with isoflurane, mice were administered an AAV vector (1 x 10 GC / mouse) for expressing human FAS via retroorbital injection. 100 μl of AAV (in PBS) was injected into the venous sinus, and the mice were monitored for recovery in their cages. Two weeks after AAV administration, the siRNA agent sets in Tables 4A and 4B were administered subcutaneously to mice as shown in Tables 6A and 6B, except that all siRNA agents lacked phosphate addition to the 5' end of the antisense strand for administration to mice.

[0191] Mice were sacrificed, serum and liver (in RNAlater Stabilization Solution, Ambion) were collected, and total liver RNA was isolated, purified, and subjected to QRT-PCR as described above.

[0192] Results show gene expression of human FAS target genes normalized to mouse Rplp0 (Life Technologies, part number: Mm01974474_gH) and expressed as relative knockdown of human FAS mRNA expression compared to vehicle-treated control animals. For the RNAi agents shown in Tables 6A and 6B, knockdown results are shown after 2 weeks of treatment at 5 mg / kg (mpk) or after 10 weeks at 1 mg / kg, 3 mg / kg, and 5 mg / kg doses.

[0193] Several RNAi agents tested for gene expression knockdown in vivo were further tested for protein knockdown according to Example 6.

[0194] [Table 5-1]

[0195] [Table 5-2]

[0196] [Table 5-3]

[0197] [Table 5-4]

[0198] [Table 5-5]

[0199] [Table 5-6]

[0200] [Table 5-7]

[0201] [Table 5-8]

[0202] [Table 5-9]

[0203] [Table 5-10]

[0204] [Table 5-11]

[0205] [Table 5-12] P indicates the 5' phosphate. m indicates a 2'O-methyl modified ribose on the listed nucleotide. f indicates a 2'F modified ribose on the listed nucleotide. * indicates a phosphorothioate bond (instead of a phosphodiester bond). GNA stands for glycol nucleic acid nucleotide. (AP) means apurinic / apyrimidinic residue, also called abasic residue.

[0206] [Table 6-1]

[0207] [Table 6-2]

[0208] [Table 6-3]

[0209] [Table 6-4]

[0210] [Table 6-5]

[0211] [Table 6-6]

[0212] [Table 6-7]

[0213] [Table 6-8]

[0214] [Table 6-9]

[0215] [Table 6-10] m indicates a 2'O-methyl modified ribose on the listed nucleotide. f indicates a 2'F modified ribose on the listed nucleotide. * indicates a phosphorothioate bond (instead of a phosphodiester bond). GNA stands for glycol nucleic acid nucleotide. (AP) means apurinic / apyrimidinic residue, also called abasic residue.

[0216] [Table 7-1]

[0217] [Table 7-2]

[0218] [Table 7-3]

[0219] [Table 7-4]

[0220] [Table 8-1]

[0221] [Table 8-2] * For all duplexes / RNAi agents tested by administration to mice, the antisense strand does not have an extra phosphate addition as shown in Table 4A.

[0222] [Table 9]

[0223] Example 6: In vivo protein quantification in AAV-Fas-expressing mice treated with RNAi agents as shown in Tables 6A and 6B. Liver samples from the RNAi-treated mice were flash-frozen and stored at -80°C. While frozen, Lysis Matrix D tubes containing approximately one-third of the liver were transferred to wet ice, and 700 μl of XYlite containing 2x Halt buffer was added to each sample at 700 μl per tube. The samples were homogenized using a Fast Prep 96 at 1800 rpm for 60 seconds and cooled on ice for 5 minutes. This process was then repeated for an additional 30 seconds, followed by a spin-down at 20,000 rcf for 5 minutes at 4°C. The samples were centrifuged in Eppendorf tubes at 20,000 rcf for 10 minutes at 4°C to remove cellular debris. Protein quantification was performed on the supernatants using the following procedure.

[0224] All samples were equilibrated to 2.0 mg / ml. The supernatant (in XY buffer) was dispensed into two 96-well plates at 100 μl per well, stored at -80°C, and subjected to protein quantification. BSA standards were prepared at 2 mg / ml and diluted with lysis buffer to create standards. Samples were diluted 1:50 by adding 2 μL of lysate to 98 μl of XY lite and HALT in a 96-well plate (Corning #3790) and mixed by pipetting. Next, 3 mL of Biorad Reagent A and 60 μl of Biorad Reagent S were combined to create Reagent C. 25 μl of Reagent C was added to each well of a 96-well plate (Corning #3596). 5 μL of standard or diluted sample was then added to each well of the 96-well plate containing Reagent C and run in duplicate. Absorbance was read at 750 nm on a 77 / 3 / 350 SpectraMax.

[0225] The quantified supernatants were then subjected to ELISA using the Human FAS DuoSet ELISA protocol. The capture antibody was diluted in PBS to a working concentration (1.0 μg / ml). 100 μl was immediately added per well and incubated overnight at room temperature. The following day, plate wells were decanted and washed three times with 300 μl / well of wash buffer. The plate was blocked by adding 300 μl / well of reagent diluent to each well and incubated for 1 hour at room temperature. The wells were decanted and washed three times with 300 μl / well of 1x wash buffer, and blotted dry after the final wash. A standard curve using control FAS protein was generated by diluting it in reagent diluent to final concentrations of 4000 pg / mL, 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, or 0 pg / mL. Thawed liver lysate or standard solution was added per well, the plate was sealed, and the plate was incubated for 2 hours at room temperature with gentle shaking. Samples were added at 100 μl / well at 0.1 μg / μl for 10 μg total protein / well diluted in reagent diluent. After incubation, the assay plate was decanted, washed three times with 300 μl / well of 1x wash buffer, and blotted dry. 100 μL / well of detection antibody diluted in reagent diluent was added. The plate was sealed and incubated for 2 hours at room temperature. The detection antibody was diluted to a working concentration of 50 ng / mL in reagent diluent. After incubation, the assay plate was decanted, washed three times with 300 μl / well of 1x wash buffer, and blotted dry. 100 μL / well of a working dilution of Strep-HRP (1:200) diluted in reagent diluent was added to the plate. The plate was covered and incubated for 20 minutes at room temperature, protected from direct light. After incubation, the assay plates were decanted, washed three times, and blotted dry. 100 μl / well of substrate solution was then added, and the plates were incubated at room temperature for 20 minutes, protected from direct light. Stop solution (50 μL) was added to each well and mixed gently.The OD of each well was measured at 77 / 3 / 350 at 450 nM using SpectraMax within 30 minutes of adding the stop solution and corrected at 540 nM (OD at 450 nM - OD at 540 nM).

[0226] The results are shown in Tables 6A and 6B, respectively.

[0227] Example 7: Additional RNAi agents tested for knockdown An additional FAS-GalNAc RNAi agent, D-235, shown in Table 7 below, was tested in vitro in HepG2 cells as described above and demonstrated approximately 50% or greater knockdown compared to vehicle controls. The RNAi agents were tested in AAV-hFAS-treated mice for mRNA knockdown and protein knockdown as described above.

[0228] [Table 10]

[0229] An RNAi agent against mouse FAS mRNA was also generated and tested (see Table 8).

[0230] [Table 11]

[0231] Example 8. Characterization of FAS RNAi agents in cynomolgus monkeys In vivo testing of selected FAS RNAi agents in cynomolgus monkeys (Macaca fascicularis) was performed to evaluate their efficacy in silencing target genes in the liver. Cynomolgus monkeys (n=3 / group) received a single subcutaneous dose of FAS RNAi agent (3 mg / kg, 0.5 ml / kg in sterile 1x PBS (pH 7.2)) or sterile 1x PBS (pH 7.2) (0.5 ml / kg). After administration of the FAS RNAi agent, wedge biopsies of the liver were collected 28 days after administration. cDNA was prepared from RNA isolated from the monkey liver samples, and qPCR was performed to measure FAS mRNA knockdown. Table 9 shows the mRNA knockdown of FAS expression in the liver 28 days after administration of the FAS RNAi agent compared to the PBS control group.

[0232] [Table 12]

Claims

1. 1. An RNAi agent for reducing FAS gene expression, the RNAi agent comprising a compound of formula I conjugated to R 【Chemistry 1】 wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand, R is conjugated to connection point E of Formula I, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, the antisense strand comprises a region of complementarity to the FAS mRNA target sequence of SEQ ID NO: 1, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages. RNAi agents.

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

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

4. The RNAi agent of any one of claims 1 to 3, wherein the antisense strand is 15 to 50 nucleotides in length.

5. The RNAi agent of any one of claims 1 to 4, wherein the sense strand is 15 to 50 nucleotides in length.

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

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

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

9. The RNAi agent of any one of claims 1 to 8, wherein the complementary region is at least 18 nucleotides in length.

10. The RNAi agent of any one of claims 1 to 9, wherein the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 2 to 112.

11. The RNAi agent of any one of claims 1 to 10, wherein the antisense strand comprises at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 224-334, 337, 338, 573 and 577.

12. The RNAi agent of any one of claims 1 to 11, wherein the antisense strand has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 224-334, 337, 338, 573 and 577, or a sequence having at least 90% sequence identity thereto.

13. The RNAi agent of any one of claims 1 to 12, wherein the sense strand is selected from the group consisting of SEQ ID NOs: 113 to 223, 335, 336, 572 and 576, or a sequence having at least 90% sequence identity thereto.

14. 14. The RNAi agent of any one of claims 1 to 13, wherein the duplex region between the sense strand and the antisense strand comprises 0, 1, 2, or 3 mismatches between the sense strand and the antisense strand.

15. the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence being a. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 129 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 240; b. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 116 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 227; c. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 151 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 262; d. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 128 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 239; and e. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 155 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

266. The RNAi agent of any one of claims 1 to 14, selected from the group consisting of:

16. the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence being a. the first nucleic acid sequence comprising SEQ ID NO: 129 and the second nucleic acid sequence comprising SEQ ID NO: 240; b. the first nucleic acid sequence comprising SEQ ID NO: 116 and the second nucleic acid sequence comprising SEQ ID NO: 227; c. the first nucleic acid sequence comprising SEQ ID NO: 151 and the second nucleic acid sequence comprising SEQ ID NO: 262; d. the first nucleic acid sequence comprising SEQ ID NO: 128 and the second nucleic acid sequence comprising SEQ ID NO: 239; and e. the first nucleic acid sequence comprising SEQ ID NO: 155 and the second nucleic acid sequence comprising SEQ ID NO: 266 The RNAi agent of any one of claims 1 to 15, selected from the group consisting of:

17. The RNAi agent of any one of claims 1 to 16, wherein the sense strand or the antisense strand each independently comprises one or more modified nucleotides.

18. 18. The RNAi agent of any one of claims 1 to 17, wherein the sense strand or the antisense strand each independently comprises one or more modified nucleotides, and the modified nucleotides are independently a 2' fluoro-modified nucleotide residue, a 2'-O-methyl-modified nucleotide, or a glycol nucleic acid (GNA) nucleotide.

19. 19. The RNAi agent of any one of claims 1 to 18, wherein the sense strand comprises one or more modified nucleotide residues, and at least one modified nucleotide residue is a GNA nucleotide present at an internal position of the sense strand.

20. The RNAi agent of any one of claims 1 to 19, wherein each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide.

21. The antisense strand is 23 nucleotides in length, and each nucleotide of the antisense strand is a modified nucleotide, and the 2' fluoro-modified nucleotide is 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 The RNAi agent of any one of claims 1 to 20, wherein

22. 22. The RNAi agent of any one of claims 1 to 21, wherein the sense strand and the antisense strand each independently comprise one or more modified internucleotide linkages, and each modified internucleotide linkage is a phosphorothioate linkage.

23. The RNAi agent of any one of claims 1 to 22, wherein the sense strand and the antisense strand each independently comprise four phosphorothioate linkages.

24. The RNAi agent of any one of claims 1 to 23, wherein the 5'-terminal nucleotide of the antisense strand comprises a phosphate group or a phosphate analog.

25. The antisense strand is selected from the group consisting of SEQ ID NOs: 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 41 0, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488 , 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 563, 566, 567, 569, 570, 571, 575, 579, 581, 583, 585, or a sequence having at least 90% sequence identity thereto, wherein the 5' terminal nucleotide of the antisense strand comprises a vinyl phosphonate, a phosphate, or a hydroxyl group.

26. The sense strand is selected from the group consisting of SEQ ID NOs: 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 09, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 48 5, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561 564, 565, 568, 574, 578, 580, 582, 584, or a sequence having at least 90% sequence identity thereto, wherein the 5'-terminal nucleotide of the antisense strand comprises a vinyl phosphonate, a phosphate, or a hydroxyl group.

27. The antisense strand is selected from the group consisting of SEQ ID NOs: 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 813, 815, 817, 819, or a sequence with at least 90% sequence identity thereto.

28. The sense strand is selected from the group consisting of SEQ ID NOs: 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650 , 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 72 28. The RNAi agent of any one of claims 1-24 or 27, comprising a sequence selected from the group consisting of: 0, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 809, 810, 811, 812, 814, 816, 818, or a sequence with at least 90% sequence identity to said sequence.

29. the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence being a. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:339 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:340; b. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:341 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:342; c. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:343 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:344; d. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:345 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:346; e. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 347 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 348; f. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 349 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 350; g. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:353 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:354; h. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 363 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 364; and i. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:381 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

382. The RNAi agent of any one of claims 1 to 28, selected from the group consisting of:

30. the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence being a. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 564 or 809 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 571; b. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 568 or 811 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 567; c. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 580 or 814 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 581 or 815; d. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 582 or 816 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 583 or 817; and e. the first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 584 or 818 and the second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 585 or 819. The RNAi agent of any one of claims 1 to 28, selected from the group consisting of:

31. the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence being a. the first nucleic acid sequence comprising SEQ ID NO: 564 or 809 and the second nucleic acid sequence comprising SEQ ID NO: 571; b. the first nucleic acid sequence comprising SEQ ID NO: 568 or 811 and the second nucleic acid sequence comprising SEQ ID NO: 567; c. the first nucleic acid sequence comprising SEQ ID NO: 580 or 814 and the second nucleic acid sequence comprising SEQ ID NO: 581 or 815; d. the first nucleic acid sequence comprising SEQ ID NO: 582 or 816 and the second nucleic acid sequence comprising SEQ ID NO: 583 or 817; and e. the first nucleic acid sequence comprising SEQ ID NO: 584 or 818 and the second nucleic acid sequence comprising SEQ ID NO: 585 or 819 The RNAi agent of any one of claims 1 to 28, selected from the group consisting of:

32. The RNAi agent of any one of claims 29 to 31, wherein the 5' terminal nucleotide of the antisense strand contains a vinyl phosphonate, a phosphate group, or an OH group.

33. 33. The RNAi agent of any one of claims 1 to 32, wherein R is conjugated to formula I via a linker.

34. R is conjugated to Formula I via a linker, wherein the linker comprises Formula II having connection points A and B, or the linker comprises Formula III having connection points C and D; 【Chemistry 2】 a. Formula I is conjugated to Formula II at connection point A, and Formula II is conjugated to a phosphate group or a phosphorothioate group at connection point B, said phosphate group or said phosphorothioate group being further conjugated to R; or b. Formula I is conjugated to Formula III at connection point C, and Formula III is conjugated to a phosphate group or a phosphorothioate group at connection point D, wherein the phosphate group or the phosphorothioate group is further conjugated to R; The RNAi agent according to any one of claims 1 to 33.

35. R is conjugated to Formula I via a linker, said linker being Formula III having connection points C and D. 【Transformation 3】 wherein Formula I is conjugated to Formula III at connection point C, and Formula III is conjugated to a phosphate group or a phosphorothioate group at connection point D, said phosphate group or said phosphorothioate group being further conjugated to R. The RNAi agent according to any one of claims 1 to 34.

36. The RNAi agent of any one of claims 1 to 35, wherein the RNAi agent reduces expression of the FAS gene in liver cells compared to a control.

37. 36. The RNAi agent of any one of claims 1 to 35 for use in therapy.

38. The RNAi agent of any one of claims 1 to 35 for use in the treatment of autoimmune hepatitis (AIH).

39. 36. A pharmaceutical composition comprising the RNAi agent of any one of claims 1 to 35 and one or more pharmaceutically acceptable excipients.

40. 36. Use of the RNAi agent of any one of claims 1 to 35 in the manufacture of a medicament for the treatment of autoimmune hepatitis (AIH).

41. 36. A method of treating autoimmune hepatitis (AIH) in a patient in need thereof, comprising administering to said patient the RNAi agent or pharmaceutical composition thereof of any one of claims 1 to 35.

42. 36. A method of reducing FAS expression in a cell, comprising contacting the cell with an RNAi agent of any one of claims 1 to 35.

43. 43. The method of claim 42, further comprising incubating the cells for a time sufficient to reduce the level of FAS mRNA by at least 50% compared to untreated or control-treated cells.

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