Novel FAS RNAi therapeutics and uses thereof

Abstract

The present invention relates to novel therapeutic compounds, referred to as RNAi agents, which reduce the expression of FAS receptors (expressed by the FAS gene), thereby reducing the expression of FAS mRNA and protein expression. Such RNAi agents are useful in the treatment of diseases involving modulation of FAS expression and function, such as autoimmune hepatitis.

Description

BACKGROUND

[0002] The present invention relates to novel therapeutic compounds, called RNAi agents, that reduce expression of FAS (expressed from the FAS gene), thereby reducing expression of FAS mRNA and FAS protein. Such RNAi agents are useful in treating diseases involving modulation of FAS expression and function, for example autoimmune hepatitis.

[0003] FAS, the Fas cell 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., caspases 8 and 10) and Fas-associated death domain protein (FADD) that form a complex. Autoproteolysis of the caspases in the complex leads to a caspase cascade and results in apoptosis. NF-KB, MAPK3 / ERK1, and MAPK8 / JNK are also known to be activated by FAS signaling, and such activation is thought to lead to proliferation in normal diploid fibroblasts and T cells. These play an important role in the modulation of immune responses involving cells expressing FAS, which include hepatocytes.

[0004] Autoimmune hepatitis (AIH) is a chronic inflammation of the liver without an identifiable cause, such as a viral infection. AIH patients have increased levels of FAS in hepatocytes. Genetics, environment (e.g., environmental triggers), and dysregulation of the innate immune system are thought to play a role in the progression of the disease from inflammation to liver fibrosis. AIH often presents for the first time in patients when they reach their teenage years.

[0005] Treatment options are limited and include high-dose steroid therapy often combined with another immunosuppressant, azathioprine. Treatment is associated with downregulation of FAS, and patients can achieve near remission of biochemical inflammation. However, steroid therapy, especially when taken long-term and / or at high doses, can cause a wide range of serious side effects, as do other immunosuppressant treatments such as azathioprine. Serious side effects can include onset of diabetes, osteoporosis (osteoporosis), bone fractures (osteonecrosis), hypertension, cataracts, glaucoma, and weight gain. If treatment is stopped, patients often experience a relapse, and some patients experience disease progression requiring liver transplantation. Accordingly, there is a need for improved treatments for AIH. SUMMARY

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

[0008]

[0009] Wherein R is optionally conjugated to the attachment point E of Formula I via a linker, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises a region having complementarity with the FAS mRNA target sequence of SEQ ID NO: 1, and wherein the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide bonds. In some embodiments, Formula I is optionally conjugated to the sense strand via a linker. In some embodiments, Formula I is optionally conjugated to the 3' terminal nucleotide of the sense strand via a linker.

[0010] In some embodiments, the antisense strand is 15 to 50 nucleotides in length. In some embodiments, the sense strand is 15 to 50 nucleotides in length. In some embodiments, the antisense strand is 18 to 23 nucleotides in length. In some embodiments, the sense strand is 18 to 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

[0011] In some embodiments, the sense strand or the antisense strand comprises a sequence selected from Table 2, 3A, 3B, 4A, 4B, 7, or 8 disclosed herein. In some embodiments, the sense strand and the antisense strand comprise a sequence selected from Table 2, 3A, 3B, 4A, 4B, 7, or 8 disclosed herein.

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

[0013]

[0014]

[0015] a. Formula I is conjugated to Formula II at connection point A and Formula II is conjugated to a phosphate group at connection point B, and the phosphate group is further conjugated to R; or

[0016] 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, and the phosphate group is further conjugated to R.

[0017] In another aspect, provided herein are pharmaceutical compositions comprising a FAS RNAi agent described herein and one or more pharmaceutically acceptable excipients.

[0018] 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 described herein, or a pharmaceutical composition thereof.

[0019] In another aspect, provided herein is a FAS RNAi agent for use in therapy. Also provided herein is a FAS RNAi agent for use in treating AIH. Also provided herein is use of a FAS RNAi agent in the manufacture of a medicament for treating AIH. DETAILED DESCRIPTION

[0021] FAS siRNAs and ASOs have been described, but none have advanced in therapy in patients, including for AIH. The use of FAS RNAi agents described herein to reduce expression of FAS can be used to treat AIH in a patient in need thereof. For example, such siRNAs can exhibit one or more of the following: improved knockdown in the liver; improved tissue exposure; improved exposure in hepatocytes; improved durable response; improved pharmacokinetic profile; less off-target effects; and / or improved toxicity profile compared to other siRNAs targeting the liver, e.g., FAS siRNAs comprising different delivery ligands, different sequences, different modified sequences, or compared to treatment with vehicle control. Other embodiments of FAS RNAi agents described herein can include one or more of the following: fewer side effects compared to steroids or other standard of care; improved toxicity profile; improved safety profile; improved tolerability or compliance; and / or improved liver function tests. Still other siRNAs herein can have other benefits, e.g., in combination with any of the foregoing or as independent benefits, including improved and / or simplified synthesis, synthesis process with fewer degradation products; or any combination thereof.

[0022] RNAi agents herein comprise a sense strand and an antisense strand, wherein each strand is an oligonucleotide. In some embodiments, RNAi agents described herein further comprise a delivery moiety. As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., such as adenine, cytosine, guanine, thymine, or uracil; and a pentose sugar, e.g., such as ribose or 2’-deoxyribose) and a phosphate group. “Nucleotides” can serve as monomeric units of nucleic acid polymers, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0023] As used herein, “oligonucleotide” means a short nucleic acid compound (e.g., less than about 100 nucleotides in length). An oligonucleotide can be single-stranded (ss) or double-stranded (ds). An oligonucleotide can or can not have duplex regions. As a non-limiting example, an oligonucleotide can be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (DsiRNA), or an antisense oligonucleotide (ASO).

[0024] As used herein, “ribonucleotide” means a nucleotide having ribose as its pentose sugar, which contains a hydroxyl group at its 2’ position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms at the 2’ position other than hydrogen, including modifications or substitutions in the nucleobase, sugar, or phosphate groups.

[0025] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared to a reference internucleotide linkage having a phosphodiester linkage. A modified internucleotide linkage can be a non-naturally occurring linkage.

[0026] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared to a corresponding reference nucleotide selected from the group consisting of: an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, an adenine deoxyribonucleotide, a guanine deoxyribonucleotide, a cytosine deoxyribonucleotide, and a thymidine deoxyribonucleotide. A modified nucleotide can be a non-naturally occurring nucleotide. A modified nucleotide can have, for example, one or more chemical modifications in its sugar, nucleobase, and / or phosphate groups. Additionally or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0027] The term "percent sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of the nucleotides, nucleosides or nucleobases identical with those in the reference nucleic acid sequence, nucleosides or nucleobases, in the candidate sequence, after the sequence is optimally aligned and, if necessary, room or overhang is introduced to achieve maximum percent sequence identity. The comparison for determining the purpose of percent nucleic acid sequence identity can be achieved in the various ways within the art, such as using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., ed., 1987, Supplement 30, section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, ClustalW2.0 or Clustal X2.0 or Megalign (DNASTAR) software. In one embodiment herein, Clustal W2.0 or Clustal X2.0 are used to calculate sequence identity. In another embodiment, Clustal W2.0 is used to calculate sequence identity. In another embodiment, use Clustal X2.0 to calculate sequence identity.Those skilled in the art can determine the appropriate parameters for measuring comparison, be included in and realize any algorithm required for maximum comparison on the total length of compared sequences.The per-cent of " sequence identity " can be determined by comparing the sequence of two optimal comparisons on a comparison window, wherein the nucleotide sequence fragment in the comparison window may comprise the interpolation or disappearance (for example, room or overhang) compared with a reference sequence (it does not comprise interpolation or disappearance), for the optimal comparison of two sequences.Percentage can be calculated by following: determine the number of positions that identical Nucleotide, nucleoside or core base occur in two sequences therein, to draw the matching position number, the matching position number is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to draw the per-cent of sequence identity.Output is the per-cent identity of subject sequence with respect to query sequence.In some embodiments, per-cent sequence identity is to use PID3 to calculate the per-cent of identical nucleotide residues between two chains, and it is the number of identical nucleotide residues divided by the total number of nucleotides of the shortest sequence in two sequences, multiplied by 100. See, eg, Raghava, G., Barton, GJ Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006).

[0028] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5’ terminal nucleotide of an oligonucleotide in place of a 5’-phosphate. A 5’ phosphate analog can include a phosphatase resistant linkage. Examples of phosphate analogs include, but are not limited to, 5’ phosphonates, such as 5’-methylene phosphonate (5’-MP) and 5’-(E)-vinyl phosphonate (5’-VP). An oligonucleotide can have a phosphate analog at the 4’ carbon position of the sugar at the 5’ terminal nucleotide (referred to as a “4’-phosphate analog”). An example of a 4’-phosphate analog is an oxymethylene phosphonate, in which the oxygen atom of the oxymethylene group is bound to a sugar moiety (e.g., at its 4’ carbon) or an analog thereof. See, e.g., International Patent Application Publication No. WO 2018 / 045317. Other modifications for the 5’ end of an oligonucleotide have been developed (see, e.g., International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0029] As used herein, “region of complementarity” means a nucleotide sequence of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to allow hybridization between the two nucleotide sequences under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotides herein include a targeting sequence having a region of complementarity to an mRNA target sequence.

[0030] As used herein, reference to a “duplex” of a nucleic acid or oligonucleotide, e.g., a sense strand or an antisense strand, means a structure formed by hydrogen bonding between complementary bases of two antiparallel nucleotide sequences, under suitable conditions to promote such structure. A duplex can form even though there is not perfect complementarity between the two strands, or when there are abasic nucleotides.

[0031] RNA interference is a specialized cellular process that utilizes RISC for the sequence-dependent degradation of RNA. As used herein, “RNAi agent” means an agent that comprises (a) a double-stranded oligonucleotide having a sense strand (passenger strand) and an antisense strand (guide strand), wherein the antisense strand or a portion of the antisense strand is used by an Argonaute 2 (Ago2) endonuclease to cleave a target mRNA, or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand (or a portion of that antisense strand) is used by an Ago2 endonuclease to cleave a target mRNA. In some embodiments, the RNAi agents described herein further comprise a delivery moiety.

[0032] As used herein, "treatment" or "treating" means all processes, whether of not indicated for complete elimination of all symptoms of a disorder or disease, wherein there can be a slowing, control, delay or stopping of the progression of a disorder or disease disclosed herein, or amelioration of symptoms of the disorder or disease. Treatment includes administration of an RNAi agent or pharmaceutical composition thereof for treating a disease or condition in a mammal, including a human.

[0033] "Effective amount" means the amount necessary to achieve the desired therapeutic result for the period of time and means of administration. The effective amount of an RNAi agent can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the RNAi agent to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the RNAi agent are outweighed by the therapeutically beneficial effects.

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

[0035]

[0036] wherein R is conjugated to the point of attachment E of Formula I via a linker, wherein the sense and antisense strands form a duplex region, and wherein the antisense strand comprises a region of complementarity to a FAS mRNA target sequence of SEQ ID NO: 1, and wherein the sense and antisense strands each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages.

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

[0038]

[0039] wherein R is conjugated to Formula la via a linker, wherein the sense and antisense strands form a duplex region, and wherein the antisense strand comprises a region of complementarity to a FAS mRNA target sequence of SEQ ID NO: 1, and wherein the sense and antisense strands each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages.

[0040] Disclosed herein are RNAi agents for reducing FAS gene expression, wherein the RNAi agent comprises a dsRNA comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand form a duplex region, and wherein the antisense strand comprises a region having at least 15 nucleotides of complementarity to a sequence as shown in SEQ ID NO: 1, and wherein the sense strand and / or antisense strand each optionally comprises one or more modified nucleotides and / or modified internucleotide linkages. In further embodiments, the antisense strand comprises at least 15 nucleotides of a sequence in Table 2. In further embodiments, the antisense strand comprises at least 18 nucleotides of a sequence in Table 2. In further embodiments, the RNAi agent reduces FAS gene expression in FAS-expressing cells by about 50% or more compared to a control. In further embodiments, the RNAi agent reduces FAS gene expression by reducing the level of FAS mRNA transcripts, the level of FAS protein, or both.

[0041] In further embodiments, the antisense strand is 15 to 50 nucleotides in length, and / or the sense strand is 15 to 50 nucleotides in length. In further embodiments, the sense strand and / or the sense strand are independently 15 to 30 nucleotides in length. In further embodiments, the antisense strand is 18 to 23 nucleotides in length. In further embodiments, the sense strand is 18 to 21 nucleotides in length.

[0042] In a further embodiment, the RNAi agent comprises an antisense strand comprising at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 2- 112. In yet a further embodiment, the antisense strand comprises at least 18 contiguous nucleotides of a sequence selected from SEQ ID NOs: 2-112.

[0043] In other further embodiments, the antisense strand comprises at least 18 contiguous nucleotides of a sequence selected from SEQ ID NOs: 224 to 334, 337, 338, 573, and 577.

[0044] In other further embodiments, the sense strand comprises at least 18 contiguous nucleotides of a sequence selected from SEQ ID NOs: 113 to 223, 335, 336, 572, and 576.

[0045] In a further embodiment, the antisense strand of the RNAi agent is 23 nucleotides in length. In yet 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 sequence shown in Table 3A.

[0046] The sense and antisense strands of the RNAi agents disclosed herein are not required to be fully complementary. Accordingly, in the RNAi agents disclosed herein, the duplex region between the sense and antisense strands comprises 0, 1, 2, or 3 mismatches between the sense and antisense strands. In further embodiments, the duplex region between the sense and antisense strands consists of 0, 1, 2, or 3 mismatches between the sense and antisense strands.

[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 selected from the group consisting of:

[0048] a. The first nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 129, and the second nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 240;

[0049] b. The first nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 116, and the second nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 227;

[0050] c. the first nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 151, and the second nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 262;

[0051] d. the first nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 128, and the second nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 239; and

[0052] e. The first nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 155, and the second nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 266.

[0053] 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 selected from the group consisting of:

[0054] a. The first nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 129, and the second nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 240;

[0055] b. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 116, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 227;

[0056] c. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 151, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 262;

[0057] d. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 128, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 239; and

[0058] e. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 155, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 266.

[0059] 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 selected from the following:

[0060] a. the first nucleic acid sequence comprises SEQ ID NO: 129, and the second nucleic acid sequence comprises SEQ ID NO: 240;

[0061] b. the first nucleic acid sequence comprises SEQ ID NO: 116, and the second nucleic acid sequence comprises SEQ ID NO: 227;

[0062] c. the first nucleic acid sequence comprises SEQ ID NO: 151, and the second nucleic acid sequence comprises SEQ ID NO: 262;

[0063] d. the first nucleic acid sequence comprises SEQ ID NO: 128, and the second nucleic acid sequence comprises SEQ ID NO: 239; and

[0064] e. the first nucleic acid sequence comprises SEQ ID NO: 155, and the second nucleic acid sequence comprises SEQ ID NO: 266.

[0065] In further embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides, such as 2' fluoro-modified nucleotides or 2'-O-methyl-modified nucleotides. In yet further embodiments of the RNAi agents disclosed herein, each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide. In further embodiments, each nucleotide is a 2' fluoro-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0066] In further embodiments of the RNAi agents disclosed herein, the antisense strand is 23 nucleotides in length, each nucleotide of the antisense strand is a modified nucleotide, and the 2' fluoro-modified nucleotide is present in

[0067] a. positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand; or

[0068] b. positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand; or

[0069] c. positions 2, 3, 8, 14, and 16 from the 5' end of the antisense strand; or

[0070] d. positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand; or

[0071] e. Positions 2, 6, 14 and 16 from the 5' end of the antisense strand.

[0072] In a further embodiment, the nucleotide that is not a 2' fluoro-modified nucleotide is a 2'-O-methyl-modified nucleotide.

[0073] In further embodiments of the RNAi agents 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 yet further embodiments, the two terminal nucleotides at each of the 5' and 3' ends of each of the sense strand and the antisense strand are phosphorothioate linkages.

[0074] In other embodiments, the 5' nucleotide of the antisense strand comprises a phosphate group or a phosphate analog. As used herein, "phosphate analog" means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is positioned at the 5' terminal nucleotide of the oligonucleotide in place of a 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). The oligonucleotide can have a phosphate analog at the 4' carbon position of the sugar at the 5' terminal nucleotide (referred to as a "4'-phosphate analog"). An example of a 4'-phosphate analog is an oxymethylene phosphonate, in which the oxygen atom of the oxymethylene group is bound to a sugar moiety (e.g., at its 4' carbon) or an analog thereof. See, e.g., International Patent Application Publication No. WO 2018 / 045317. Other modifications for the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0075] In further embodiments of the RNAi agents disclosed herein, the antisense strand comprises a sequence selected from the group consisting of SEQ ID NO: 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, 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, phosphate, or hydroxyl group. In other embodiments, the phosphate group listed at the 5' end of the SEQ ID NO: is removed and replaced with OH. In other embodiments, the phosphate group listed at the 5' end of the SEQ ID NO: is replaced with a 5' vinyl phosphonate.

[0076] In further embodiments of the RNAi agents disclosed herein, the antisense strand comprises a sequence selected from the group consisting of SEQ ID NO: 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 having at least 90% sequence identity thereto.

[0077] In further embodiments, the sense strand comprises a sequence selected from the group consisting of SEQ ID NO: 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.

[0078] In a further embodiment, the sense strand comprises a sequence selected from the group consisting of: SEQ ID NO: 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, 802, 804, 806, 808, 809, 810, 811, 812, 814, 816, 818, or a sequence thereof having at least 90% sequence identity.

[0079] In yet further embodiments of the RNAi agents disclosed herein, the sense and antisense strands are a pair of oligonucleotide sequences selected from Table 4A or 4B, or sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to a sequence in Table 4A or 4B. In further embodiments, 1, 2, or 3 mismatches are introduced into the sense strand of a pair in Table 4A, Table 4B, or Table 7. In further embodiments, 1, 2, or both terminal nucleotides at the 5' end of the antisense strand are altered.

[0080] 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 selected from the group consisting of:

[0081] a. The first nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 339, and the second nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 340;

[0082] b. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 341 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 342;

[0083] c. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 343 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 344;

[0084] d. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 345 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 346;

[0085] e. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 347 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 348;

[0086] f. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 349 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 350;

[0087] g. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 353 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 354;

[0088] h. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 363 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 364; and

[0089] i. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 381 and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 382.

[0090] 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 selected from the following:

[0091] a. the first nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 564 or 809 and the second nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 571;

[0092] b. The first nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 568 or 811, and the second nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 567;

[0093] c. the first nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 580 or 814, and the second nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 581 or 815;

[0094] d. the first nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 582 or 816, and the second nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 583 or 817; and

[0095] e. The first nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 584 or 818, and the second nucleic acid sequence has at least 90% sequence identity with SEQ ID NO: 585 or 819.

[0096] 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 selected from the group consisting of:

[0097] a. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 564 or 809, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 571;

[0098] b. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 568 or 811, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 567;

[0099] c. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 580 or 814, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 581 or 815;

[0100] d. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 582 or 816, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 583 or 817; and

[0101] e. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 584 or 818, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 585 or 819.

[0102] 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 selected from the group consisting of:

[0103] a. The first nucleic acid sequence comprises SEQ ID NO: 564 or 809, and the second nucleic acid sequence comprises SEQ ID NO: 571;

[0104] b. The first nucleic acid sequence comprises SEQ ID NO: 568 or 811, and the second nucleic acid sequence comprises SEQ ID NO: 567;

[0105] c. The first nucleic acid sequence comprises SEQ ID NO: 580 or 814, and the second nucleic acid sequence comprises SEQ ID NO: 581 or 815;

[0106] d. the first nucleic acid sequence comprises SEQ ID NO: 582 or 816, and the second nucleic acid sequence comprises SEQ ID NO: 583 or 817; and

[0107] e. The first nucleic acid sequence comprises SEQ ID NO: 584 or 818, and the second nucleic acid sequence comprises SEQ ID NO: 585 or 819.

[0108] 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 selected from the group consisting of:

[0109] a. The first nucleic acid sequence consists of SEQ ID NO: 564 or 809, and the second nucleic acid sequence consists of SEQ ID NO: 571;

[0110] b. The first nucleic acid sequence consists of SEQ ID NO: 568 or 811, and the second nucleic acid sequence consists of SEQ ID NO: 567;

[0111] c. the first nucleic acid sequence consists of SEQ ID NO: 580 or 814, and the second nucleic acid sequence consists of SEQ ID NO: 581 or 815;

[0112] d. the first nucleic acid sequence consists of SEQ ID NO: 582 or 816, and the second nucleic acid sequence consists of SEQ ID NO: 583 or 817; and

[0113] e. the first nucleic acid sequence consists of SEQ ID NO: 584 or 818, and the second nucleic acid sequence consists of SEQ ID NO: 585 or 819.

[0114] In further embodiments, the 5' terminal nucleotide of the antisense strand is substituted such that the final sequence contains a vinyl phosphonate, phosphate group, or OH group. For example, for the following antisense sequences: SEQ ID NO: 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, 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, the 5' phosphate group is replaced with an OH group.

[0115] In other embodiments, disclosed herein are RNAi agents having a delivery moiety of Formula I conjugated to R:

[0116]

[0117] wherein R is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides of complementarity to a FAS mRNA target sequence of SEQ ID NO: 1, and wherein the sense strand and the antisense strand form a region of complementarity of at least 15 nucleotides, and wherein the sense strand and the antisense strand are each independently 18 to 23 nucleotides in length, and optionally wherein the sense strand and the antisense strand each independently comprise one or more modified nucleotides, and optionally wherein the sense strand and the antisense strand each independently comprise one or more modified internucleotide linkages, and wherein R is conjugated to Formula I via a linker. In further embodiments, the sense strand or the antisense strand is selected from Table 2, 3A, 3B, 4A, 4B, 7, or 8 disclosed herein. In other embodiments, the antisense strand or the antisense strand of the RNAi agent has a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding sequence selected from Table 2, 3A, 3B, 4A, 4B, 7, or 8 herein.

[0118] 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 comprises a linker of Formula II having attachment points A and B, or the linker comprises Formula III having attachment points C and D, and wherein:

[0119]

[0120]

[0121] a. Formula I is conjugated at attachment point A to Formula II and Formula II is conjugated at attachment point B to a phosphato group, and the phosphato group is conjugated to R; or

[0122] b. Formula I is conjugated at attachment point C to Formula III and Formula III is conjugated at attachment point D to a phosphato group, and the phosphato group is further conjugated to R.

[0123] In other embodiments wherein the RNAi agent comprises a linker, R is conjugated to Formula I via a linker, and the linker is a linker comprising Formula III having attachment points C and D:

[0124]

[0125] and wherein Formula I is conjugated at attachment point C to Formula III and Formula III is conjugated at attachment point D to a phosphato group, and the phosphato group is further conjugated to R.

[0126] The sense and antisense strands of the FAS RNAi agent can be synthesized using any nucleic acid polymerization method known in the art, for example, by solid phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercial synthesizers, such as the MerMade 9 TM 12, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using sulfurizing reagents such as phenacyl disulfide or DTTT ((dimethylaminomethylene)amino)-3H-1,2,4-dithiazoline-3-thione). It is well known to use similar techniques, along with commercially available modified amidites and controlled pore glass (CPG) products, to synthesize modified oligonucleotides.

[0127] In yet other embodiments, the RNAi agent is capable of reducing expression of the FAS gene in hepatocytes. In other embodiments, the RNAi agents disclosed herein are used for therapy. In further embodiments, the use is for the treatment of AIH.

[0128] The RNAi agent can be formulated into a pharmaceutical composition. Accordingly, disclosed herein are pharmaceutical compositions comprising the RNAi agents disclosed herein and one or more pharmaceutically acceptable excipients. The pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rdEd. (2020), A. Loyd et al., Academic Press).

[0129] In other embodiments is the use of the RNAi agents herein for the manufacture of a medicament for the treatment of AIH.

[0130] In other embodiments is a method of treating AIH in a patient in need thereof comprising administering a FAS RNAi agent disclosed herein or a pharmaceutical composition thereof.

[0131] The RNAi agent can be administered intravenously or subcutaneously to the patient.

[0132] The RNAi agent dosage regimen 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.

[0133] The dosage values can vary as widely as tlie nature and severity of the condition being alleviated permit. 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.

[0134] In other embodiments are methods of reducing FAS expression in a cell, comprising contacting the cell with an RNAi agent disclosed herein, and incubating the cell for a period of time sufficient to reduce FAS mRNA levels by at least 50% as compared to untreated or control treated cells. Examples

[0135] Certain abbreviations are defined below:“1,2-DCE” refers to 1,2-dichloroethane;“DCM” refers to dichloromethane;“DIEA” refers to N,N-diisopropylethylamine;“DMF” refers to N,N-dimethylformamide;“DMAP” refers to 4-dimethylaminopyridine;“DMTCl” refers to 4,4'-dimethoxytrityl chloride;“DPP4” refers to dipeptidyl peptidase;“EDC” refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;“EtOAc” refers to ethyl acetate;“GalNAc” refers to N-acetylgalactosamine;“HATU” refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate;“HBTU” refers to O-(Benzotriazol-1-yl)-N,N,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 methylalcohol;“MWCO” refers to molecular weight cut-off;“NHS” refers to N-hydroxysuccinimide;“OD” refers to optical density;“PBS” refers to phosphate buffered saline;“PhSiH3” refers to phenylsilane;“PTS” refers to portable endotoxin test system;“siRNA” refers to small interfering ribonucleic acid;“TEA” refers to triethylamine;“TFA” refers to trifluoroacetic acid;“THF” refers to tetrahydrofuran;“TLC” refers to thin line chromatography; and“TMP” refers to 2,2,6,6-tetramethylpiperidine.

[0136] Delivery moieties comprising Formula I can be prepared by the following non-limiting synthesis steps and schemes.

[0137] Scheme 1

[0138]

[0139] Scheme 1, Step A, depicts the cyclization of compound (1) using trimethylsilyl triflate in a solvent such as 1,2-DCE to give compound (2). Step B shows the addition of hex-5-ene-1-ol to compound (2) using trimethylsilyl triflate in a solvent such as 1,2-DCE to give compound (3). Oxidation of compound (3) using an appropriate oxidizing agent such as sodium periodate with a catalyst such as ruthenium(III) chloride is shown in Step C to give compound (4).

[0140] Scheme 2

[0141]

[0142] Scheme 2, step A, shows an amide coupling between compound (5) and N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamic acid tert-butyl ester using HBTU and HOBt in a solvent such as DMF, along with a suitable base such as DIEA, to give compound (6). Step B depicts a basic hydrolysis of compound (6) using a base such as aqueous NaOH in a THF and MeOH solvent system to give compound (7). Step C shows an amide coupling between compound (7) and allyl 11-aminoundecanoate hydrochloride using HATU in a solvent such as DMF, along with a suitable base such as DIEA, to give compound (8). Step D shows an acidic deprotection of compound (8) with TFA in a solvent such as DCM to give compound (9). An amide coupling between compound (9) and compound (4) using EDC and HOBt in a solvent such as DCM is shown in step E to give compound (10). Step F shows a deprotection of compound (10) with tetrakis(triphenylphosphine)palladium and PhSiH3in a solvent such as DCM to give compound (11). Step F depicts a coupling of compound (11) with NHS using EDC in a solvent such as DCM to give compound (12).

[0143] Scheme 3

[0144]

[0145] Scheme 3, steps A-C are essentially similar to those of Scheme 2, steps C-E, starting with compound (7) to give compounds (13), (14), and (15). Step D depicts a 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 Scheme 2, step G, to give compound (17).

[0146] Scheme 4

[0147]

[0148] Scheme 4, steps A-I, consists of a series of amide couplings and deprotections, using methods essentially similar to those found in Schemes 2 and 3, starting with compound (18) to give compound (27).

[0149] Scheme 5

[0150]

[0151] Scheme 5, steps A-C depict methods essentially similar to those found in Scheme 4, steps G-I, starting with compound (24) to give compound (30).

[0152] Scheme 6

[0153]

[0154] Scheme 6, step A depicts the protection of compound (31) using DMTCl in a solvent such as DCM along with a suitable base such as DIEA to give compound (32). Step B shows an amide coupling between compound (32) and piperidin-4-ylmethanol using HBTU and HOBt along with TMP in a solvent such as DCM to give compound (33). Deprotection of compound (33) with 20% piperidine in DMF is shown in step C to give compound (34).

[0155] Scheme 7

[0156]

[0157] Scheme 7, step A is essentially similar to Scheme 2, step A, to give compound (35) from the coupling of compounds (16) and (34). Step B shows the formation of compound (36) by the addition of succinic anhydride to compound (35) in a suitable solvent such as DCM with a base system of TEA and DMAP. Step C depicts the loading of compound (36) onto a resin along with 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate and a base such as DIEA in a solvent system such as MeCN and DCM to give compound (37).

[0158] Preparation 1

[0159] (6,7-Diacetyloxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetic acid methyl ester

[0160]

[0161] To a solution of (5-acetylamino-3,4,6-triacetoxy-tetrahydropyran-2-yl)acetic acid methyl ester (9.00 g, 23.1 mmol) in 1,2-DCE (46 mL) was added trimethylsilyl triflate (6.5 mL, 35 mmol). The mixture was heated to 50 °C and stirred for 18 h. After this time, the mixture was diluted with DCM (200 mL), washed with saturated NaHCO3(200 mL) and saturated aqueous sodium chloride (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 0-10% MeOH / DCM to give the title compound (6.434 g, 84%). ES / MS m / z 330 (M+H).

[0162] Preparation 2

[0163] (5-acetylamino-3,4-diacetoxy-6-hex-5-enyloxy-tetrahydropyran-2-yl)acetic acid methyl ester

[0164]

[0165] To a solution of (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]oxazol-5-yl)acetic acid methyl ester (30.43 g, 92.42 mmol) in 1,2-DCE (231 mL) was added hex-5-ene-1-ol (22.2 mL, 185 mmol) followed by activated powdered molecular sieves (15.6 g). The suspension was stirred at ambient temperature for 30 min, then trimethylsilyl triflate (19 mL, 101.9 mmol) was added. The mixture was stirred at ambient temperature for 18 h. After this time, the solution was filtered through celite and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 30-100% EtOAc / hexanes to give the title compound (34.76 g, 86%). ES / MS m / z 430.4 (M+H).

[0166] Preparation 3

[0167] 5-[3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxypentanoic acid

[0168]

[0169] A solution of (5-acetylamino-3,4-diacetoxy-6-hex-5-enyloxy-tetrahydropyran-2- yl)acetic acid methyl ester (34.76 g, 80.93 mmol) in MeCN (174 mL) and DCM (174 mL) was cooled to 0 °C. A solution of sodium periodate (22.4 g, 104.7 mmol) was added and stirring was continued at 0 °C for 10 min. After this time, ruthenium (III) chloride (270 mg, 1.3 mmol) was added and the mixture was stirred while warming to ambient temperature. After stirring for 2 h, additional sodium periodate (66 g, 308.4 mmol) was added and stirring was continued for 18 h. After this time, the mixture was extracted with 3:1 CH3Cl:IPA (2 x 500 mL), washed with saturated aqueous sodium chloride solution (1 L), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was 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).

[0170] Preparation 4

[0171] 6-Aminohexanoic acid benzyl ester hydrochloride

[0172]

[0173] To a suspension of 6-aminohexanoic acid (5.00 g, 38.1 mmol) in THF (38 mL) was added benzyl alcohol (47 mL, 453.7 mmol) and the mixture was cooled to 0 °C. Sulfurous acid chloride (8.6 mL, 120 mmol) was added dropwise and the mixture was stirred while warming to ambient temperature for 18 h. After this time, diethyl ether (166 mL) was added and the reaction vessel was transferred to a freezer chamber at -20 °C for 1 h. After this time, the solid precipitate was collected by filtration to give the title compound (8.57 g, 81%). ES / MS m / z 222 (M+H).

[0174] Preparation 5

[0175] 11-Aminoundecanoic acid benzyl ester hydrochloride

[0176]

[0177] The title compound was prepared from 11-aminoundecanoic acid in a manner substantially similar to the method of Preparation 4. ES / MS m / z 292.2 (M+H).

[0178] Preparation 6

[0179] 11-aminoundecanoic acid allyl ester hydrochloride

[0180]

[0181] A solution of 11-aminoundecanoic acid (9.00 g, 44.7 mmol) in allyl alcohol (42 mL) was charged to a vessel and the mixture was cooled to 0 °C. Sulfoxene (6.5 mL, 89.4 mmol) was added and the mixture was stirred while warming to ambient temperature for 18 hours. After this time, the mixture was concentrated in vacuo and diethyl ether (200 mL) was added to the residue to give a white suspension. The mixture was stirred at ambient temperature for 10 minutes and the solid precipitate was collected by filtration to give the product (12.0 g, 97%). ES / MS m / z 242.2 (M+H).

[0182] Preparation 7

[0183] (2S)-3-[Bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9-ylmethoxycarbonylamino)propionic acid

[0184]

[0185] To a stirred solution of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3- hydroxy-propionic acid (40 g, 0.122 mol) in dry DCM (400 mL) at 0 °C under inert atmosphere was added DIEA (64 mL, 0.366 mol). To this was added slowly a solution of DMTCl (49.6 g, 0.146 mol) in DCM (200 mL). The resulting reaction mixture was allowed to reach ambient temperature and stirred for 16 hours. After this time, the reaction mixture was diluted with water (12.5 volumes) and extracted with DCM (25 volumes). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting crude was washed with 10% EtOAc / hexane (12.5 volumes) and dried under vacuum to give the title compound as a light brown solid (62 g, crude). This material was taken to the next step without any further purification. TLC: 5% MeOH / CH2Cl2(Rf: 0.5) UV, 254 nM.

[0186] Preparation 8

[0187] 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxo-ethyl]carbamate

[0188]

[0189] To a stirred solution of (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9- ylmethoxycarbonylamino)propionic acid (62 g, 0.103 mol) in DCM (750 mL) was added slowly HBTU (78.3 g, 0.206 mol), HOBt (27.9 g, 0.206 mol) and piperidin-4-ylmethanol (15.4 g, 0.134 mol) followed by TMP (15 mL, 0.113 mol) at 0 °C under inert atmosphere. The resulting reaction mixture was allowed to reach ambient temperature and stirred for 4 h. After this time, the reaction mixture was diluted with water (8 vol) and extracted with DCM (15 vol). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting residue was 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) δ 7.88 (br d, J = 7.5 Hz, 2H), 7.79-7.59 (m, 3H), 7.45-7.12 (m, 13H), 6.92-6.76 (m, 4H), 4.79-4.44 (m, 2H), 4.32 (br d, J = 11.4 Hz, 2H), 4.20 (br s, 2H), 3.71 (s, 6H), 3.21 (br s, 4H), 2.99-2.79 (m, 1H), 2.69 (br s, 2H), 1.81-1.43 (m, 3H), 1.08-0.73 (m, 2H).

[0190] Preparation 9

[0191] (2S)-2-Amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1- piperidinyl]propan-1-one

[0192]

[0193] A 20% solution of piperidine in DMF (400 mL) was added slowly to 9H-fluoren-9- ylmethyl N-[(1 S)-1 -[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)- 1 -piperidinyl]-2-oxo-ethyl]carbamate (40 g, 0.055 mol) at 0 °C under an inert atmosphere. The resulting reaction mixture was stirred at ambient temperature for 1 h. After this time, the mixture was diluted with water (15 vol) and extracted with EtOAc (30 vol). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography eluting with 1-8% MeOH / DCM to give the title compound as an off-white solid (13 g, 47%). ES / MS m / z 1009.5 (2M+H).

[0194] Preparation 10

[0195] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxopentanoic acid methyl ester

[0196]

[0197] 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) was added DMF (179 mL) and (2-(1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (11.7 g, 30.9 mmol). DIEA (14 mL, 80.3 mmol) was added and the mixture was stirred at ambient temperature for 5 min. After this time, a portion of tert-butyl N-[2-[2-(tert- butoxycarbonylamino)ethylamino]ethyl]carbamate (8.94 g, 29.5 mmol) was added and stirring was continued at ambient temperature. After 18 h of stirring, the mixture was diluted with EtOAc (400 mL), washed with water (2 x 400 mL) and saturated aqueous sodium chloride solution (400 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography eluting with 40-100% EtOAc / hexanes to give the title compound (13.01 g, 89%). ES / MS m / z 547.40 (M+H).

[0198] Preparation 11

[0199] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxopentanoic acid

[0200]

[0201] A flask was charged with (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]- 2-(tert-butoxycarbonylamino)-5-oxopentanoic acid methyl ester (13.01 g, 23.8 mmol), THF (120 mL), and MeOH (120 mL). IN NaOH (71 mL, 71 mmol) was added and the mixture was stirred at ambient temperature. After 1 h, the mixture was concentrated in vacuo and redissolved in water (300 mL). 5N HCl (12 mL) was added to bring the pH to 4. The mixture was extracted with DCM (3 x 300 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (1 L), dried over sodium sulfate, filtered, and concentrated to give the title compound (12.41 g, 98%). ES / MS m / z 531.60 (M-H).

[0202] Preparation 12

[0203] 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoic acid allyl ester

[0204]

[0205] To a flask containing (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2- (tert-butoxycarbonylamino)-5-oxopentanoic acid (500 mg, 0.94 mmol) and allyl 11- aminoundecanoate hydrochloride (313 mg, 1.13 mmol) was added DMF (6.25 mL) and l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (428 mg, 1.12 mmol). After addition of DIEA (0.5 mL, 3 mmol), the mixture was stirred at ambient temperature for 18 h. After this time, the mixture was 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 was purified by silica gel flash chromatography eluting with 40-100% EtOAc / hexanes to give the title compound (687 mg, 97%).1H NMR (DMSO-d6) δ 7.78-7.64 (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).

[0206] Preparation 13

[0207] (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)undecanoic acid allyl ester

[0208]

[0209] To a solution of 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoic acid allyl ester (687 mg, 0.91 mmol) in DCM (15 mL) was added TFA (15 mL). The mixture was stirred at ambient temperature. After 1.5 hours, the mixture was concentrated in vacuo. The residue was taken up in MeOH and applied to an ion exchange cartridge. The cartridge was eluted with MeOH (150 mL) followed by 7 N NH3 / MeOH (150 mL). The basic fractions were concentrated in vacuo to give the title compound (410 mg, 99%). ES / MS m / z 456.4 (M+H).

[0210] Preparation 14

[0211] 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5- oxo-pentanoyl]amino]undecanoic acid allyl ester

[0212]

[0213] A flask was charged with 5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxy pentanoic acid (489 mg, 1.09 mmol) and (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)undecanoic acid allyl ester (150 mg, 0.33 mmol). DCM (3.35 mL) was added followed by 1-hydroxybenzotriazole monohydrate (164 mg, 1.07 mmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (206 mg, 1.07 mmol). The mixture was stirred at ambient temperature for 18 hours. After this time, the solution was diluted with EtOAc (100 mL) and washed with saturated NaHCO3(2 x 100 mL), saturated aqueous NH4Cl (100 mL), and saturated aqueous sodium chloride (100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 0-10% MeOH / DCM to give the title compound (424 mg, 74%). ES / MS m / z 872.80 (M+2H) / 2.

[0214] Preparation 15

[0215] 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]ethyl]amino]-5- oxo-pentanoyl]amino]undecanoic acid

[0216]

[0217] To a solution of 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]-5-[bis[2-[5-[3- acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvaleryl- amino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid allyl ester (354 mg, 0.20 mmol) in DCM (2 mL) was added tetrakis(triphenylphosphine)palladium (29 mg, 0.02 mmol) followed by PhSiH3(51 uL, 0.41 mmol). The mixture was stirred at ambient temperature for 2 h after which it was diluted with saturated aqueous NaHCO3(100 mL). IN NaOH (15 mL) was added to bring the pH to ~10. The aqueous solution was washed with DCM (3 x 100 mL) then acidified with concentrated HCl (5 mL) followed by 5 N aqueous HCl (15 mL). The aqueous layer was extracted with DCM (100 mL) and the organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was 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.

[0218] Preparation 16

[0219] (2,5-dioxopyrrolidin-1-yl) 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]-5-[bis[2-[5-[3- acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvaleryl- amino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate

[0220]

[0221] To a reaction vial was added 11-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxy-pentanoylamino]-5-[bis[2-[5-[3- acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxy- pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid (50 mg, 0.03 mmol), N-hydroxysuccinimide (5 mg, 0.04 mmol) and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (8 mg, 0.04 mmol). DCM (0.3 mL) was added and the mixture was stirred at ambient temperature. After 18 hours the mixture was loaded directly onto a silica gel cartridge and the crude mixture was purified by flash chromatography on silica gel eluting with 0-10% MeOH / DCM to give the title compound (49 mg, 93%). ES / MS m / z 901.40 (M+2H) / 2.

[0222] Preparation 17

[0223] 6-[[(2S)-5-[Bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoic acid

[0224]

[0225] The title compound was prepared from (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid and 6-aminohexanoic acid benzyl ester hydrochloride in a manner substantially similar to the procedure for the preparation of 10. ES / MS m / z 736.40 (M+H).

[0226] Preparation 18

[0227] 6-[[(2S)-2-Amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoic acid benzyl ester tris(trifluoroacetate)

[0228]

[0229] To a solution of 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoic acid benzyl ester (15.47 g, 21.02 mmol) in DCM (105 mL) was added TFA (16 mL, 210.2 mmol). The mixture was stirred at ambient temperature for 24 hours. After this time, additional TFA (16 mL, 210.2 mmol) was added and stirring was continued for a further 2 hours. After this time, the mixture was concentrated in vacuo. The resulting residue was azeotroped with toluene (2 x 30 mL). The resulting oil was further dried in a vacuum oven at 40 °C for 4 hours to give the title compound (28.08 g, 58% purity calculated for residual toluene, 99+%) ES / MS m / z 436.40 (M+H). The compound was dissolved in 70 mL DMF to make a 0.3 M solution for use in the next step.

[0230] Preparation 19

[0231] 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5- oxo-pentanoyl]amino]hexanoic acid benzyl ester

[0232]

[0233] The title compound was prepared from 5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxy pentanoic acid and 6-[[(2S)-2-amino-5- bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoic acid benzyl ester trifluoroacetic acid in a manner essentially similar to the method of preparation 10. ES / MS m / z 862 (M+2H) / 2.

[0234] Preparation 20

[0235] 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5- oxo-pentanoyl]amino]hexanoic acid

[0236]

[0237] Palladium on carbon (1.90 g, 0.89 mmol, 5 mass%, 50% wet weight) was placed in a round bottom flask and the vessel was evacuated and backfilled with nitrogen three times. A solution of 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-5-[bis[2-[5-[3-acetylamino-4,5- diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolethyl]amino]ethyl]amino]- 5-oxo-pentanoyl]amino]hexanoic acid benzyl ester (15.41 g, 8.94 mmol) in MeOH (178 mL) was added via syringe. The flask was evacuated and backfilled with 1 atm of hydrogen and the mixture was stirred at ambient temperature under 1 atm of hydrogen for 18 hours. After this time, the mixture was filtered through celite and the filtrate was concentrated in vacuo to give the title compound (13.85 g, 95%). ES / MS m / z 817.2 (M+2H) / 2.

[0238] Preparation 21

[0239] (2,5-dioxopyrrolidin-l-yl) 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-5-[bis[2-[5-[3-acetylamino-4,5- diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolethyl]amino]ethyl]amino]- 5-oxo-pentanoyl]amino]hexanoate

[0240]

[0241] The title compound was prepared from 6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-5-[bis[2-[5-[3-acetylamino-4,5- diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolethyl]amino]ethyl]amino]- 5-oxo-pentanoyl]amino]hexanoic acid in a manner essentially similar to that described for the preparation of 16. ES / MS m / z 866.20 (M+2H) / 2.

[0242] Preparation 22

[0243] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert- butoxycarbonylamino)-5-oxo-pentanoic acid benzyl ester

[0244]

[0245] The title compound was prepared from tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate and (4S)-5-benzyloxy-4-(tert-butoxycarbonylamino)-5- oxopentanoic acid in a manner substantially similar to the procedure for preparing 12. ES / MS m / z 623.6 (M+H).

[0246] Preparation 23

[0247] (2S)-2-Amino-5-[bis(2-aminoethylamino]-5-oxo-pentanoic acid benzyl ester tris(trifluoroacetate) salt

[0248]

[0249] The title compound was prepared from (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5- oxo-pentanoic acid benzyl ester in a manner substantially similar to the procedure for preparing 18. ES / MS m / z 620.4 (M+H).

[0250] Preparation 24

[0251] (2S)-5-[Bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert- butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoic acid benzyl ester

[0252]

[0253] The title compound was prepared from 5-(tert-butoxycarbonylamino)pentanoic acid and (2S)-2-amino-5-[bis(2-aminoethylamino]-5-oxo-pentanoic acid benzyl ester tris(trifluoroacetate) salt in a manner substantially similar to the procedure for preparing 10. ES / MS m / z 920.6 (M+H).

[0254] Preparation 25

[0255] (2S)-2-(5-Aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5- oxo-pentanoic acid benzyl ester tris(trifluoroacetate) salt

[0256]

[0257] The title compound was prepared from (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5- oxo-pentanoic acid benzyl ester in a manner substantially similar to the procedure for preparing 18. ES / MS m / z 620.4 (M+H).

[0258] Preparation 26

[0259] (2S)-2-[5-[5-[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]oxyvalerylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoylamino]ethyl]amino]- 5-oxo-pentanoic acid

[0260]

[0261] The title compound is prepared from 5-[3-acetylamino-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]oxyvaleric acid and (2S)-2-(5-aminopentanoyl- amino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoic acid benzyl ester tris(trifluoroacetate) salt in a manner essentially similar to that described for the preparation of 10. ES / MS m / z 954.80 (M+2H) / 2.

[0262] Preparation 27

[0263] (2S)-2-[5-[5-[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]oxyvalerylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoylamino]ethyl]amino]- 5-oxo-pentanoic acid

[0264]

[0265] Palladium on carbon (467 mg, 0.22 mmol, 5 mass%, 50% wet weight) was charged to a round bottom flask and the flask was evacuated and backfilled with nitrogen three times. A solution of (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoic acid benzyl ester (4.19 g, 2.20 mmol) in MeOH (44 mL) was added via syringe followed by three drops of acetic acid. The flask was evacuated and backfilled with 1 atm of hydrogen and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After 2 hours, the mixture was filtered through celite and the filtrate was concentrated in vacuo to give the title compound (3.99 g, 99+%). ES / MS m / z 909.6 (M+2H) / 2.

[0266] Preparation 28

[0267] 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoic acid benzyl ester (4.19 g, 2.20 mmol) in MeOH (44 mL) was added via syringe followed by three drops of acetic acid. The flask was evacuated and backfilled with 1 atm of hydrogen and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After 2 hours, the mixture was filtered through celite and the filtrate was concentrated in vacuo to give the title compound (3.99 g, 99+%). ES / MS m / z 909.6 (M+2H) / 2.

[0268]

[0269] The title compound was prepared from (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoic acid and 6- aminohexanoic acid benzyl ester hydrochloride in a manner essentially similar to the method of Preparation 10. ES / MS m / z 1011.6 (M+2H) / 2.

[0270] Preparation 29

[0271] 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoylamino]-5- [bis[2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid

[0272]

[0273] A round bottom flask was charged with palladium on carbon (24 mg, 0.01 mmol, 5% by weight, 50% wet weight) and the flask was evacuated and backfilled with nitrogen. A solution of 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoylamino]-5- [bis[2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid benzyl ester (222 mg, 0.11 mmol) in MeOH (2.2 mL) followed by three drops of acetic acid was added via syringe. The flask was evacuated and backfilled with 1 atm of hydrogen and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After 5 hours, the flask was purged with nitrogen and the mixture was filtered through celite. The filtrate was concentrated in vacuo to give the title compound (180 mg, 85%). ES / MS m / z 966.2 (M+2H) / 2.

[0274] Preparation 30

[0275] (2,5-dioxopyrrolidin-1-yl) 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoylamino]-5- [bis[2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0276]

[0277] The title compound was prepared from 6-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid in a manner essentially similar to the method for preparing 16. ES / MS m / z 1014.6 (M+2H) / 2.

[0278] Preparation 31

[0279] Benzyl 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate

[0280]

[0281] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 11-aminoundecanoate hydrochloride in a manner essentially similar to the method for preparing 10. ES / MS m / z 1046.6 (M+2H) / 2.

[0282] Preparation 32

[0283] 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid

[0284]

[0285] To a round bottom flask was added palladium on carbon (35 mg, 0.02 mmol, 5 mass%, 50% wet weight) and the flask was evacuated and backfilled with nitrogen three times. A solution of 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid benzyl ester (285 mg, 80% purity, 0.11 mmol) was added via syringe. The vessel was evacuated and backfilled with 1 atm of hydrogen and the mixture was then stirred at ambient temperature under 1 atm of hydrogen. After 3 hours of stirring, the flask was purged with nitrogen and the mixture was filtered through celite. The filtrate was concentrated to give the title compound (213 mg, 79% purity, 77%). ES / MS m / z 1001.20 (M+2H) / 2.

[0286] Preparation 33

[0287] (2,5-dioxopyrrolidin-l-yl) 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid ester

[0288]

[0289] The title compound was prepared from 11-[[(2S)-2-[5-[5-[3-acetylamino-4,5- diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol- 5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid in a manner essentially similar to that described for the preparation of 16. ES / MS m / z 1050 (M+2H) / 2

[0290] Preparation 34

[0291] Methyl 5-acetamido-6-[5-[2-[[(4S)-4-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-5-[[6-[[(1S)-1-[[bis(4- methoxyphenyl)-phenylmethoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2- oxoethyl]amino]-6-oxohexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-ethyl]amino] ethylamino]-5-oxapentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]acetate

[0292]

[0293] The title compound is prepared from 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-5-[bis[2-[5-[3-acetamido- 4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-ethyl]amino] ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid and (2S)-2-amino-3-[bis(4- methoxyphenyl)-phenylmethoxy]-1-[4-(hydroxymethyl)-1-piperidinyl]propan-1-one in a manner essentially similar to that used for the preparation of 10. ES / MS m / z 1059.2 (M-2H) / 2.

[0294] Preparation 35

[0295] 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-5-[bis[2-[5-[3-acetamido- 4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol-ethyl]amino] ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)- phenylmethoxy]propanoyl]-4-piperidinyl]methoxy]-4-oxobutanoic acid

[0296]

[0297] To a solution of methyl [5-acetylamino-6-[5-[2-[[(4S)-4-[5-[3-acetylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxy-pentanoylamino]-5-[[6-[[(1S)-1- [[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2- oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxy-pentanoylamino]ethyl]amino]- 5-oxo-pentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]acetate (1.194 g, 0.56 mmol) in DCM (11 mL) was added succinic anhydride (113 mg, 1.13 mmol), TEA (0.4 mL, 3 mmol) and DMAP (213 mg, 1.69 mmol). The mixture was stirred at ambient temperature for 1 hour. After this time, the mixture was diluted with saturated NH4Cl (200 mL) and extracted with DCM (3 x 200 mL) and 3:1 CHCl3:IPA (200 mL). The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography eluting with 0-40% MeOH / DCM and the resulting product was dried in a vacuum oven at 40 °C for 3 hours to give the title compound (1.081 g, 86%). ES / MS m / z 1109.60 (M-2H) / 2.

[0298] Preparation 36

[0299] Resin loading

[0300]

[0301] A solution of 4-[[l-[(2S)-2-[6-[[(2S)-2-[5-[3-acetylamino-4,5-diacetyloxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxy pentanoylamino]-5-[bis[2-[5-[3- acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxy pentanoyl- amino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)- phenylmethoxy]propionyl]-4-piperidinyl]methoxy]-4-oxobutanoic acid (1.00 g, 0.61 mmol) in MeCN (6 mL) and DCM (1 mL) was transferred to a resin loading syringe. To the vessel was added 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (386 mg, 0.97 mmol) and DIEA (0.25 mL, 0.48 mmol) and the syringe shaken at ambient temperature for 5 minutes. After this time, a solution of 2-mercaptoacetic acid (0.05 mL, 0.61 mmol) in MeCN (1 mL) was added and the syringe shaken at ambient temperature for 18 hours. After this time, the syringe was emptied by suction and the resin washed by shaking with DCM (10 mL) for 10 minutes. The syringe was emptied and the wash and emptying procedure repeated with 10% MeOH / DCM (10 mL) and Et2O (10 mL). After emptying, the resin was dried under vacuum for 30 minutes. The resin loading was determined using a standard trityl assay. The resin loading was calculated to be 34.7 μmol / g. LCAA controlled pore glass resin (5.39 g, 90 μmol / g loading, purchased from ChemGenes) was added and the mixture shaken at ambient temperature for 18 hours. After this time, the syringe was emptied by suction and the resin washed by shaking with DCM (10 mL) for 10 minutes. The syringe was emptied and the wash and emptying procedure repeated with 10% MeOH / DCM (10 mL) and Et2O (10 mL). After emptying, a solution of acetic anhydride (6.4 mL), pyridine (20 mL) and TEA (0.22 mL) was added and the syringe shaken for 2 hours. After this time, the syringe was emptied and the wash and emptying procedure repeated with DCM (10 mL), 10% MeOH / DCM (10 mL) and diethyl ether (10 mL). After emptying, the resin was dried under vacuum for 30 minutes. The resin loading was determined using a standard trityl assay. The resin loading was calculated to be 34.7 μmol / g.

[0302] Preparation 37

[0303] 2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydropyran- 2-yl]oxy pentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetylamino-4,5- diacetyloxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxy pentanoylamino]pentanoyl- amino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethyl acetate

[0304]

[0305] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]ethyl]amino]-5-oxo-pentanoic acid and 2-(2-aminoethoxy)acetic acid benzyl ester hydrochloride in a manner essentially similar to the procedure for the preparation of 10. ES / MS m / z 1005.2 (M+2H / 2).

[0306] Preparation 38

[0307] 2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetic acid

[0308]

[0309] Benzyl 2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetic acid (0.120 mmol, 240 mg) was combined with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 mL). The mixture was hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 48 minutes, filtered through celite, and concentrated in vacuo to give the title compound as a grey solid (187 mg, 82%). ES / MS m / z 960.0 (M+2H / 2).

[0310] Preparation 39

[0311] (2,3,5,6-tetrafluorophenyl)2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy] ethyl acetate

[0312]

[0313] To a solution of 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy] ethyl acetate (0.096 mmol, 184 mg) and DIEA (0.765 mmol, 140 μί) in DCM (3.0 mL) was added (2,3,5,6-tetrafluorophenyl)2,2,2-trifluoroacetate (0.383 mmol, 100 mg) dropwise. The mixture was stirred at ambient temperature for 16 h. The reaction mixture was purified directly by flash chromatography on silica gel 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).

[0314] Preparation 40

[0315] Benzyl 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoylaminol-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2- yl]oxyvalerylaminolpentanoylaminol]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy] ethoxy]ethoxy]ethoxy]acetate

[0316]

[0317] The title compound was prepared from (2S)-2-[5-[5-[3-acetylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl- amino]-5-[bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetra- hydropyran-2-yl]oxyvalerylaminolpentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetic acid benzyl ester hydrochloride in a manner substantially similar to the procedure for preparing 10. ES / MS m / z 1049.0 (M+2H / 2).

[0318] Preparation 41

[0319] 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetra- hydropyran-2-yl]oxyvalerylaminolpentanoyl-amino]-5-[bis[2-[5-[5-[3-acetylamino- 4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminol- pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid

[0320]

[0321] Benzyl 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylaminolpentanoyl-amino]-5- [bis[2-[5-[5-[3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydropyran- 2-yl]oxyvalerylaminolpentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino] ethoxy]ethoxy]ethoxy]ethanoate (0.118 mmol, 247 mg) was combined with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 mL). The mixture was hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 1 hour, filtered through celite, and concentrated in vacuo to give the title compound as a grey solid (227 mg, 96%). ES / MS m / z 1004.0 (M+2H / 2).

[0322] Preparation 42

[0323] (2,3,5,6-tetrafluorophenyl)2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoyl- amino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahy- dro- pyran-2-yl]oxyvalerylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino] ethoxy]ethoxy]ethoxy]acetate

[0324]

[0325] To a solution of 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydropyran-2-yl]oxyvalerylamino]pentanoylamino]-5- [bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahy- dro- pyran-2-yl]oxyvalerylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino] ethoxy]ethoxy]ethoxy]acetate (0.111 mmol, 222 mg) and DIEA (0.883 mmol, 154 μί) in DCM (3.0 mL) was added (2,3,5,6-tetrafluorophenyl)2,2,2-trifluoroacetate (0.443 mmol, 116 mg) dropwise. The mixture was stirred at ambient temperature for 16 h. The reaction mixture was directly purified by flash chromatography on silica gel 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).

[0326] Example 1: Conjugation Scheme

[0327] To synthesize the GalNAc conjugated sense strand, the sense strand was first synthesized with a 3’ C6-NH2 functional group using standard phosphoramidite chemistry. A stock solution of the GalNAc ligand-NHS ester was prepared (10 mmol / L in acetonitrile; 1 equivalent). Phosphate buffer (10% v / v; 20x) was added to the oligonucleotide C6-NH2 sense strand in an Eppendorf tube, followed by the GalNAc ligand (5 equivalents). The mixture was allowed to shake at ambient temperature for 16 hours. Following this, the mixture was transferred to a 15 mL Falcon tube, ammonium hydroxide (28 mass%) was added, and the mixture was allowed to shake 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 NaH2P04, Solvent B: 15% MeCN / 20 mM NaH2P04, 1 M NaBr; 35% - 55% B over 5 CV at 8 mL / min, column temperature 60 °C. The desired fractions were combined and desalted by spin filtration using an Eppendorf centrifuge or a desalting column. Following desalting, the material was recovered and the OD and volume were measured to obtain the concentration.

[0328] Alternatively, conjugation to the 5’ position of the sense strand was performed by immobilising the GalNAc ligand to a microporous polystyrene resin or controlled pore glass and performing synthesis with 5’-CE beta-cyanoethyl) phosphoramidites using established solid phase oligonucleotide synthesis methods.

[0329] Alternatively, the GalNAc ligand was converted to a suitable phosphoramidite and delivered to the 5’ position of the sense strand using standard phosphoramidite chemistry.

[0330] Example 2: Annealing

[0331] To generate siRNA duplexes of the sense and antisense strands, the following procedure was performed. To a Falcon tube containing the oligonucleotide sense-GalNAc conjugate, the corresponding antisense oligonucleotide was added (1 equivalent) and vortexed for 10 seconds, followed by spin filtration through a 100 K MWCO Amicon filter device to remove particulates. The filtrate was recovered and concentrated in vacuo on a Genevac evaporator. The residue was reconstituted in 1x PBS, filtered through a 0.2 μ filter, and the OD and volume were measured to obtain the concentration.

[0332] Endotoxin testing was performed using Limulus amoebocyte lysate Endotoxin testing was performed using Limulus amoebocyte lysate

[0333] Table 1 - Exemplary molecules synthesized using the conjugation and annealing protocols described above.

[0334]

[0335] Example 3:

[0336] General procedure for oligonucleotide synthesis using GalNAc-functionalized CPG

[0337] Using phosphoramidite chemistry in MerMade TM Oligonucleotide synthesis was performed on a 12-well instrument. The sense strand was synthesized on a pre-functionalized GalNAc solid support, and the antisense strand was synthesized using a standard support preloaded with the first nucleotide of the oligonucleotide sequence. The oligonucleotides were cleaved and deprotected using a concentrated ammonium hydroxide solution (28% by mass) and purified by ion exchange chromatography using the above-mentioned conditions. Desalting, annealing, and endotoxin testing were performed.

[0338] The sequence of the antisense oligonucleotide was designed using 15 to 50 nucleotides of the following FAS transcript (SEQ ID NO: 1), wherein T nucleotides are replaced by U nucleotides, and wherein one or more nucleotides and one or more internucleotide linkages are optionally further modified as described herein.

[0339] Homo sapiens FAS cell death receptor (FAS) transcript,

[0340] SEQ ID NO: 1

[0341]

[0342]

[0343] Exemplary antisense strand sequences of 18 nucleotides in length are shown below in Table 2, which sequences may optionally be further modified and synthesized and incorporated into RNAi agents as described herein.

[0344] Table 2 Antisense 18-mers of FAS RNAi agents

[0345]

[0346]

[0347]

[0348] Table 3A - Exemplary full-length sense and antisense strands of FAS RNAi agents

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] GNA refers to a glycol nucleic acid nucleotide (structure shown in Table 3B);

[0359] (AP) means an apurinic / apyrimidinic residue, also known as an abasic residue (structure shown in Table 3B).

[0360] Table 3B: Structures of GNA and abasic residues

[0361]

[0362] Example 4:

[0363] In vitro knockdown of hFAS in HepG2 cells

[0364] The RNAi reagents in Tables 4A and 4B were tested in HepG2 cells. Reverse transfection was performed by adding 24.7 μΐ of Opti-MEM plus 0.3 μΐ of Lipofectamine RNAiMAX / well to each well of a collagen type I coated 96-well plate. The mixture was incubated at room temperature for 20 minutes, then 50 μΐ of growth media containing HepG2 cells at 300,000 cells / mL was added to the human FAS-GalNAc siRNA / RNAiMAX mixture. The final concentration of siRNA was 500 nM for single concentration screens as above. Cells were incubated for 24-48 hours followed by RNA isolation with the Quick-RNA 96 kit. RNA was then stored at -80 °C or subjected to cDNA synthesis. Briefly, cDNA was synthesized from purified RNA using the Fast Advanced RT Master Mix (Invitrogen). A master mix was prepared of 5 μΐ 2X Fast Advanced RT Buffer and 0.5 μΐ 20X Fast Advanced RT Enzyme Mix per reaction. 5.5 μΐ of master mix and 4.5 μΐ of RNA were mixed for a final volume of 10 μΐ. The cDNA was generated using a ProFlex PCR System (Life Technologies) by the following steps: 37 °C for 30 minutes, 95 °C for 5 minutes, and 4 °C hold.

[0365] Two μΐ of cDNA was added to a master mix containing 2.5 μΐ of H20, 0.5 μΐ 20X TaqMan Gene Expression Assay Buffer (Life Technologies), and 5 μΐ 2X TaqMan Universal PCR Master Mix (Life Technologies). A QuantStudio 7 Flex Real-Time PCR System (Life Technologies) was used to complete the following PCR cycles: 50 °C for 2 minutes, 95 °C for 10 minutes, 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. TaqMan Gene Expression Assays were performed. Data analysis used the ddCt method.

[0366] The siRNAs selected from each assay for determination of IC50 were used in a 1 :3 serial dilution to a final concentration of 200, 67, 22, 7.41, 2.47, 0.82, and 0.27 nM FAS-GalNAc RNAi reagent for concentration response curves. IC50 values were calculated using XLFit using a 4 parameter fit model.

[0367] Data is shown in Table 5.

[0368] Example 5:

[0369] In vivo knockdown in hFAS-AAV treated mice with FAS RNAi agents herein

[0370] Mice were administered an AAV vector for expression of human FAS (1 x 1011 GC / mouse) via retro-orbital injection after anesthesia via isoflurane. 100 ul of AAV (in PBS) was injected into the sinus venosus and the mice were monitored for recovery in the cage. Two weeks after AAV administration, the mice were administered a set of siRNA reagents in Tables 4A and 4B, as indicated in Tables 6A and 6B, except that all siRNA reagents for administration to the mice lacked a phosphate addition on the 5' end of the antisense strand.

[0371] Mice were sacrificed and serum and livers (in RNAlater Stabilization Solution, Ambion) were collected. Total liver RNA was isolated, purified, and subjected to QRT-PCR as described above.

[0372] Results show that gene expression of the human FAS target gene was normalized to mouse RplpO (Life Technologies, Part #: Mm01974474_gH) and is expressed as relative knockdown of human FAS mRNA expression compared to vehicle treated control animals. Knockdown results are shown for 2 weeks after treatment at 5 mg / kg (mpk) or 10 weeks after treatment at 1 mg / kg, 3 mg / kg, and 5 mg / kg doses for the RNAi reagents indicated in Tables 6A and 6B.

[0373] Some of the RNAi reagents tested for in vivo gene expression knockdown were further tested for protein knockdown in accordance with Example 6.

[0374] Table 4A - FAS-GalNAc RNAi agents, modified sense and antisense strands

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] P indicates 5' phosphate;

[0389] m indicates 2' O-methyl modified ribose on the listed nucleotide;

[0390] f indicates 2' F modified ribose on the listed nucleotide;

[0391] * indicates phosphorothioate linkage (instead of phosphodiester linkage);

[0392] GNA indicates glycol nucleic acid nucleotide;

[0393] (AP) means an abasic residue, also known as an apurinic / apyrimidinic residue.

[0394] Table 4B - FAS-GalNAc RNAi agents, modified sense and antisense strands

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408] m indicates a 2' O-methyl modified ribose on the listed nucleotide;

[0409] f indicates a 2'F modified ribose on the listed nucleotide;

[0410] * indicates a phosphorothioate linkage (instead of a phosphodiester linkage);

[0411] GNA indicates a glycol nucleic acid nucleotide;

[0412] (AP) means an apurinic / apyrimidinic residue, also known as an abasic residue.

[0413] Table 5: Percent inhibition of human FAS expression in HepG2 cells

[0414]

[0415]

[0416]

[0417]

[0418] Table 6A: In vivo FAS mRNA knockdown (%KD) and remaining FAS protein in hFAS- expressing mice treated with RNAi agents Table 6B. In vivo FAS mRNA knockdown (%KD) in hFAS-expressing mice treated with RNAi agents

[0419]

[0420]

[0421] * For all duplexes / RNAi agents tested via administration to mice, no additional phosphates were added to the antisense strand, as shown in Table 4A.

[0422] Example 6:

[0423]

[0424] In vivo protein knockdown in AAV-Fas expressing mice treated with RNAi agents as shown in Tables 6A and 6B.

[0425] Example 7: Additional RNAi agents for knockdown testing Table 7: Additional sequences

[0426] Liver samples from mice treated with the above RNAi reagents were snap frozen and stored at -80°C. While frozen, lysate D tubes containing ~1 / 3 of the liver were transferred to wet ice and XY lite containing 2X Halt buffer was added to each sample at 700ul / tube. Samples were homogenized using a Fast Prep 96 at 1800rpm for 60 seconds and cooled on ice for 5 minutes. The process was then repeated for another round of homogenization for 30 seconds followed by spinning down at 20,000rcf for 5 minutes at 4°C. Samples were centrifuged at 20,000rcf for 10 minutes in Eppendorf tubes at 4°C to remove cell debris. Protein quantification was performed on the supernatant using the following procedure.

[0427] All samples were equilibrated to 2.0mg / ml. Supernatants (in XY buffer) were aliquoted into 2 blocks of 96 well plates at 100ul sample / well and stored at -80°C and subjected to protein quantification. BSA standards were prepared at 2mg / ml and diluted in lysis buffer to make standards. Samples were diluted 1:50 by adding 2ul lysate to 98ul XY lite and HALT in a 96 well plate (Corning #3790) and mixed by pipetting. Next, 3mL Biorad reagent A and 60ul Biorad reagent S were combined to make reagent C. 25ul of reagent C was added to each well of a 96 well plate (Corning #3596). Next, 5uL of standard or diluted sample was added to each well of the 96 well plate containing reagent C and performed in duplicate. Absorbance at 750nm was read on a SpectraMax at 77 / 3 / 350.

[0428] The quantified supernatant was then subjected to ELISA using the Human FAS DuoSet ELISA protocol. The capture antibody was diluted to working concentration (1.0 ug / ml) in PBS. 100 ul / well was added immediately and incubated overnight at room temperature. The next day, the plate wells were decanted and washed 3 times with 300 uL / well of wash buffer. The plate was blocked by adding 300 ul / well of reagent diluent to each well and incubated for 1 hour at room temperature. The wells were decanted and washed 3 times with 300 ul / well of IX wash buffer and aspirated after the final wash. A standard curve with control FAS protein was prepared by diluting to a final concentration of 4000, 2000, 1000, 500, 250, 125, 62.5, or 0 pg / ml in reagent diluent. The thawed liver lysate or standards were added per well and the plate was sealed and incubated with gentle shaking for 2 hours at room temperature. Samples were added at 100 ul / well for 10 ug total protein / well diluted in reagent diluent at 0.1 ug / ul. After incubation, the assay plate was decanted and washed 3 times with 300 ul / well of IX wash buffer and aspirated. 100 ul / 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 with reagent diluent. After incubation, the assay plate was decanted and washed 3 times with 300 ul / well of IX wash buffer and aspirated. 100 ul / well of 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 plate was decanted, washed 3 times and aspirated. Next, 100 ul / well of substrate solution was added and the plate was incubated for 20 minutes at room temperature, protected from direct light. A stop solution (50 uL) was added to each well and mixed gently. Within 30 minutes after the addition of the stop solution, the OD of each well was measured at 450 nm with a correction at 540 nm (OD@450 nm - OD@540 nm) using a SpectraMax at 77 / 3 / 350.

[0429] Results are shown in Tables 6A and 6B.

[0430] Table 8: RNAi agents against mouse FAS mRNA

[0431] Additional FAS-GalNAc RNAi Reagent D-235 shown below in Table 7 was tested in vitro in HepG2 cells as described above and showed about 50% or greater knockdown compared to vehicle control. The RNAi Reagent was tested for mRNA and protein knockdown in AAV-hFAS treated mice as described above.

[0432] Table 9: FAS mRNA knockdown in cynomolgus monkeys.

[0433]

[0434] RNAi reagents against mouse FAS mRNA were also generated and tested (see Table 8).

[0435] Table 9: FAS mRNA knockdown in cynomolgus monkeys.

[0436]

[0437] Example 8. Characterization of FAS RNAi reagents in Cynomolgus monkeys

[0438] In vivo testing of selected FAS RNAi reagents in Cynomolgus monkeys (Macaca fascicularis) was performed to evaluate their efficacy in silencing the target gene in the liver. Cynomolgus monkeys (n=3 / group) were administered a single subcutaneous administration of FAS RNAi reagents (3 mg / kg in sterile lx PBS, pH 7.2, 0.5 ml / kg) or sterile lx PBS, pH 7.2 (0.5 ml / kg). Following administration of the FAS RNAi reagents, open wedge biopsies of the liver were collected 28 days post administration. cDNA was prepared from RNA isolated from the monkey liver samples and qPCR was performed to determine FAS mRNA knockdown. Table 9 shows the mRNA knockdown of FAS expression in the liver 28 days post administration of the FAS RNAi reagents compared to the PBS control group.

[0439] ​

[0440]

Claims

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

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

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

4. The RNAi agent according to 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 region of complementarity 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 SEQ ID NO: 2 to SEQ ID NO:

112.

11. The RNAi agent of any one of claims 1 to 10, wherein the antisense strand comprises at least 18 contiguous nucleotides of a sequence selected from SEQ ID NOs: 224 to 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 SEQ ID NOs: 113 to 223, 335, 336, 572, and 576, or a sequence having at least 90% sequence identity thereto.

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 RNAi agent of any one of claims 1 to 14, wherein the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are selected from the following: a. The first nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 129, and the second nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 240; b. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 116, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 227; c. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 151, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 262; d. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 128, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 239; and e. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 155, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:

266.

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

266.

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

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

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

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

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

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

23. The RNAi agent of any one of claims 1 to 22, wherein the sense strand and 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 RNAi agent of any one of claims 1 to 24, wherein the antisense strand comprises a sequence selected from the group consisting of: SEQ ID NO: 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, 407, 408, 411, 412, 413, 414, 415 06, 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 4, 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 vinylphosphonate, a phosphate, or a hydroxyl group.

26. The RNAi agent of any one of claims 1 to 25, wherein the sense strand comprises a sequence selected from the group consisting of SEQ ID NO: 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, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415 3. 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, 53 7, 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 vinylphosphonate, a phosphate, or a hydroxyl group.

27. The RNAi agent of any one of claims 1 to 24, wherein the antisense strand comprises a sequence selected from the group consisting of: SEQ ID NO:587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 6 15, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 6 45, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 6 75, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 70 79, 791, 793, 795, 797, 799, 801, 803, 805, 807, 813, 815, 817, 819, or a sequence thereof having at least 90% sequence identity.

28. The RNAi agent of any one of claims 1 to 24 or 27, wherein the sense strand comprises a sequence selected from the group consisting of SEQ ID NO: 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, 802, 804, 806, 808, 809, 810, 811, 812, 814, 816, 818, or a sequence thereof having at least 90% sequence identity.

29. The RNAi agent of any one of claims 1 to 28, wherein the sense strand comprises a first nucleic acid sequence and the antisense strand comprises a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are selected from the group consisting of: a. The first nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 339, and the second nucleic acid sequence has at least 95% sequence identity with SEQ ID NO: 340; b. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 341, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 342; c. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 343, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 344; d. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 345, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 346; e. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 347, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 348; f. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 349, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 350; g. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 353, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 354; h. the first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 363, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 364; and i. The first nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 381, and the second nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:

382.

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

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

32. The RNAi agent of any one of claims 29-31, wherein the 5' terminal nucleotide of the antisense strand comprises a vinylphosphonate, a phosphate group, or an OH group.

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

34. The RNAi agent of claims 1 to 33, wherein R is conjugated to Formula I via a linker, and wherein the linker comprises a linker of Formula II having attachment points A and B, or the linker comprises Formula III having attachment points C and D, and wherein: a. Formula I is conjugated to Formula II at the connection point A and Formula II is conjugated to a phosphate or phosphorothioate group at the connection point B, and the phosphate or phosphorothioate group is further conjugated to R; or b. Formula I is conjugated to Formula III at the connection point C and Formula III is conjugated to a phosphate or phosphorothioate group at the connection point D, and the phosphate or phosphorothioate group is further conjugated to R.

35. The RNAi agent of any one of claims 1 to 34, wherein R is conjugated to Formula I via a linker, and wherein the linker is a linker comprising Formula III with attachment points C and D: And 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, and the phosphate group or the phosphorothioate group is further conjugated to R.

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

37. 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 treating autoimmune hepatitis (AIH).

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

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

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

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

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

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