RNAi construct and method for inhibiting MARC1 expression

Abstract

The present invention relates to an RNAi construct for reducing the expression of the MARC1 gene.Methods of using such RNAi constructs to treat or prevent liver fibrosis and fatty liver disease, such as non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, are also described.The present invention is based in part on the design and generation of an RNAi construct that targets the MARC1 gene and reduces its expression in liver cells.Sequence-specific inhibition of MARC1 gene expression is useful for treating or preventing pathologies associated with high lipid levels and liver fat, such as cardiovascular disease and fatty liver disease.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 065,190 filed on 13 August 2020 and U.S. Provisional Patent Application No. 63 / 214,016 filed on 23 June 2021, both of which are incorporated herein by reference in their entirety.

[0002] Description of the electronically submitted text file This application includes a sequence listing, electronically filed in ASCII format and incorporated in its entirety herein by reference. A computer-readable copy of the sequence listing, prepared on 3 August 2021, is named A-2664-WO-PCT_ST25 and is 1,064 kilobytes in size.

[0003] The present invention relates to compositions and methods for regulating the expression of mitochondrial amidoxime reducing component 1 (mARC1) protein in the liver. Specifically, the present invention relates to nucleic acid-based therapeutics for reducing MARC1 gene expression via RNA interference, and to methods for using such nucleic acid-based therapeutics to lower circulating lipid levels and to treat or prevent fatty liver disease and hepatic fibrosis. [Background technology]

[0004] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, encompassing a variety of liver pathologies. Its prevalence has doubled in the last 20 years, and it is now estimated that approximately 20-30% of the world's population is affected. In some individuals, ectopic fat accumulation in the liver, known as steatosis, triggers inflammation and hepatocyte damage, leading to a more advanced stage of the disease called non-alcoholic steatohepatitis (NASH). NASH is defined as lipid accumulation accompanied by evidence of cell damage, inflammation, and varying degrees of scarring or fibrosis. As of 2015, an estimated 75-100 million Americans had NAFLD, while NASH accounted for approximately 10-30% of NAFLD diagnoses.The mARC1 protein is a molybdenum-containing protein in the outer mitochondrial membrane that catalyzes the reduction of N-oxidized molecules (Klein et al., J Biol Chem, Vol.287(51):42795-42803, 2012; Ott et al., J Biol Inorg Chem, Vol.20(2):265-275, 2015). It is a highly effective counterpart to CYP450, one of the most excellent endogenous converting enzymes, and is involved in the activation of amidoxime prodrugs and the deactivation of other drugs containing N-hydroxylated functional groups (Neve et al., PLoS One, Vol.10(9):e0138487, 2015; Ott et al., 2015 (above)). Recently, predicted loss-of-function variants in the MARC1 gene have been reported to be associated with lower blood levels of cholesterol and liver enzymes, reduction of hepatic fat, and prevention of cirrhosis. See Emdin et al., bioRxiv 594523; / / doi.org / 10.1101 / 594523,2019; and Emdin et al., PLoS Genet, Vol.16(4):e1008629,2020. In particular, the A165T missense variant in the mARC1 coding region was associated with the prevention of cirrhosis of all causes, a reduction in hepatic fat levels on computed tomography, a reduction in the probability of fatty liver as diagnosed by a physician, and a reduction in blood levels of alanine transaminase, alkaline phosphatase, total cholesterol, and LDL cholesterol in an analysis of 12,361 cases of cirrhosis of all causes and 790,095 controls from eight cohorts (Emdin et al.,2020 (see above)). Additional MARC1 alleles (M187K missense mutation and R200Ter truncation mutation) associated with decreased cholesterol and liver enzyme levels and a reduced risk of cirrhosis were also identified (Emdin et al., 2020 (see above)). These data suggest that deficiency of the mARC1 enzyme prevents chronic liver disease and cirrhosis.Therefore, therapeutic agents targeting the mARC1 function represent a novel approach for reducing cholesterol levels (e.g., non-HDL cholesterol or LDL cholesterol levels), liver fibrosis, and treating or preventing liver diseases, particularly NAFLD and NASH.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0006] The present invention is partially based on the design and generation of RNAi constructs that target the MARC1 gene and reduce its expression in hepatocytes. Sequence-specific inhibition of MARC1 gene expression is useful for treating or preventing conditions associated with high lipid levels and hepatic fat, such as cardiovascular disease and fatty liver disease. Accordingly, in one embodiment, the present invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence substantially complementary to the mRNA sequence of mARC1. For example, in some embodiments, the antisense strand comprises a sequence substantially complementary to a sequence of at least 15 consecutive nucleotides from the region of the human mARC1 mRNA sequence (SEQ ID NO: 1), with a mismatch of 1, 2, or 3 or fewer. In certain embodiments, the antisense strand comprises a region having at least 15 consecutive nucleotides from the antisense sequences listed in Table 1 or Table 2.

[0007] In some embodiments, the sense strand of the RNAi construct described herein contains a sequence that is sufficiently complementary to the sequence of the antisense strand to form a double-stranded region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense strand and the antisense strand are each independently about 19 to about 30 nucleotides in length. In some embodiments, the RNAi construct contains one or two blunt ends. In other embodiments, the RNAi construct contains one or two nucleotide overhangs. Such nucleotide overhangs may contain 1 to 6 unpaired nucleotides and may be located at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' ends of both the sense and antisense strands. In certain embodiments, the RNAi construct contains two unpaired nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct contains two unpaired nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 3' end of the sense strand / 5' end of the antisense strand.

[0008] The RNAi construct of the present invention may comprise one or more modified nucleotides, including nucleotides having modifications to a ribose ring, a nucleic acid base, or a phosphodiester skeleton. In some embodiments, the RNAi construct comprises one or more 2'-modified nucleotides. Such 2'-modified nucleotides may include 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), deoxyribonucleotides, or combinations thereof. In a particular embodiment, the RNAi construct comprises one or more 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, or combinations thereof. In some embodiments, all nucleotides in the sense and antisense strands of the RNAi construct are modified nucleotides. The RNAi construct of the present invention may incorporate debasalized nucleotides as terminal nucleotides, for example, at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand. In such embodiments, the debasalized nucleotide may be inverted and may be bound to an adjacent nucleotide, for example, via a 3'-3' nucleotide bond or a 5'-5' nucleotide bond.

[0009] In some embodiments, the RNAi construct includes at least one skeletal modification, such as a modified nucleotide bond or a nucleoside bond. In certain embodiments, the RNAi construct described herein includes at least one phosphorothioate nucleotide bond. In certain embodiments, the phosphorothioate nucleotide bond may be located at the 3' or 5' end of the sense strand and / or antisense strand. For example, in some embodiments, the antisense strand includes two consecutive phosphorothioate nucleotide bonds between the terminal nucleotides at both the 3' and 5' ends. In some such embodiments, the sense strand includes one or two phosphorothioate nucleotide bonds between the terminal nucleotides at its 3' end.

[0010] In certain embodiments, the antisense and / or sense strands of the RNAi construct of the present invention may include or consist of antisense sequences and sequences derived from sense sequences listed in Table 1 or Table 2. In certain such embodiments, the RNAi construct may be one of the double-stranded compounds listed in any one of Tables 1 to 24. For some purposes, the RNAi construct is D-1044, D-1061, D-1062, D-1067, D-1083, D-1090, D-1092, D-1093, D-1095, D-1138, D-1139, D-1143, D-1170, D-1177, D-1180, D-1191, D-1245, D-2000, D-2002, D-2003, D-2004, D-2011, D-2026, D-2028, D-2032, D-2033, D-2 034, D-2035, D-2036, D-2042, D-2044, D-2045, D-2046, D-2050, D-2078, D-2079, D-2081, D-2182, D-2196, D-2238, D-2241, D-2 243, D-2246, D-2255, D-2356, D-2258, D-2301, D-2316, D-2317, D-2329, D-2332, D-2341, D-2344, D-2357, D-2399 or D-2510. In certain embodiments, the RNAi construct is D-2079, D-2081, D-2196, D-2238, D-2241, D-2255, D-2258, D-2317, D-2332, D-2357, or D-2399.

[0011] In some embodiments, the RNAi construct of the present invention may target a specific region of the human mARC1 mRNA transcript (e.g., the human mARC1 mRNA transcript sequence described in SEQ ID NO: 1). For example, in certain embodiments, the RNAi construct comprises a sense strand and an antisense strand, the antisense strand comprising a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1205 to 1250 of SEQ ID NO: 1. In other embodiments, the antisense strand comprising a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1209 to 1239 of SEQ ID NO: 1. In yet another embodiment, the antisense strand comprising a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1345 to 1375 of SEQ ID NO: 1. In yet another embodiment, the antisense strand comprising a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2039 to 2078 of SEQ ID NO: 1. In certain other embodiments, the antisense strand includes a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2048 to 2074 of SEQ ID NO: 1. In any of the above embodiments, the sequence of the antisense strand may be substantially complementary to the sequence of at least 15 consecutive nucleotides in a particular region of the human mARC1 transcript (SEQ ID NO: 1), and the mismatch between the sequence of the antisense strand and the sequence of the particular region of the human mARC1 transcript is 1, 2, or 3 or less. In some of these embodiments where a mismatch occurs between the sequence of the antisense strand and the sequence of the target mARC1 mRNA sequence, the mismatch may be located between the target mARC1 mRNA sequence and the nucleotides from the 5' end to positions 6 and / or 8 of the antisense strand. In other embodiments, the sequence of the antisense strand may be perfectly complementary to the sequence of at least 15 consecutive nucleotides in a particular region of the human mARC1 transcript (SEQ ID NO: 1).

[0012] The RNAi construct of the present invention may further comprise a ligand that facilitates the delivery or uptake of the RNAi construct to specific tissues or cells, such as hepatocytes. In certain embodiments, the ligand targets the delivery of the RNAi construct to hepatocytes. In these and other embodiments, the ligand may comprise galactose, galactosamine, or N-acetyl-galactosamine (GalNAc). In certain embodiments, the ligand comprises a polyvalent galactose or polyvalent GalNAc moiety, such as a trivalent or tetravalent galactose or GalNAc moiety. The ligand may optionally be covalently bonded to the 5' or 3' end of the sense strand of the RNAi construct via a linker. In some embodiments, the RNAi construct comprises a ligand and linker having one of the structures of formulas I to IX described herein. In certain embodiments, the RNAi construct comprises a ligand and linker having the structure of formula VII. In other embodiments, the RNAi construct comprises a ligand and linker having the structure of formula IV.

[0013] The present invention also provides pharmaceutical compositions comprising the RNAi construct described herein and any of a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutical compositions are particularly useful for reducing the expression of the MARC1 gene in the cells of patients (e.g., hepatocytes) where such reduction is needed. Patients who may be administered the pharmaceutical compositions of the present invention include those diagnosed with or at risk of cardiovascular disease, fatty liver disease, hepatic fibrosis, or cirrhosis, and those with high blood cholesterol levels (e.g., total cholesterol, non-HDL cholesterol, or LDL cholesterol). Accordingly, the present invention includes methods for treating, preventing, or reducing the risk of developing fatty liver disease (e.g., NAFLD, NASH, alcoholic fatty liver disease, or alcoholic steatohepatitis), hepatic fibrosis, or cardiovascular disease in patients where such treatment is needed, comprising administering the RNAi construct or pharmaceutical composition described herein. In certain embodiments, the present invention provides a method for reducing blood levels (serum or plasma) of cholesterol (e.g., total cholesterol, non-HDL cholesterol, or LDL cholesterol) in a patient in need thereof, comprising administering an RNAi construct or pharmaceutical composition as described herein.

[0014] The use of a mARC1-targeted RNAi construct for preparing a pharmaceutical for administration by any of the methods described herein or by any of the methods described herein is specifically considered. For example, the present invention includes a mARC1-targeted RNAi construct for use in a method of treating, preventing or reducing the risk of developing fatty liver disease (e.g., NAFLD, NASH, alcoholic fatty liver disease or alcoholic steatohepatitis), hepatic fibrosis or cardiovascular disease in patients who need it. The present invention also includes a mARC1-targeted RNAi construct for use in a method of reducing blood levels (serum or plasma) of cholesterol (e.g., total cholesterol, non-HDL cholesterol or LDL cholesterol) in patients who need it.

[0015] The present invention also includes the use of a mARC1-targeted RNAi construct in the preparation of a pharmaceutical for the treatment, prevention, or reduction of the risk of developing fatty liver disease (e.g., NAFLD, NASH, alcoholic fatty liver disease, or alcoholic steatohepatitis), hepatic fibrosis, or cardiovascular disease in patients in need. In certain embodiments, the present invention provides the use of a mARC1-targeted RNAi construct in the preparation of a pharmaceutical for the reduction of blood levels (serum or plasma) of cholesterol (e.g., total cholesterol, non-HDL cholesterol, or LDL cholesterol) in patients in need. In embodiments of the present invention, for example, the following items are provided. (Item 1) An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence substantially complementary to the mRNA sequence of mARC1, and the region comprises at least 15 consecutive nucleotides from the antisense sequences listed in Table 1 or Table 2. (Item 2) The RNAi construct described in item 1, wherein the sense strand contains a sequence sufficiently complementary to the sequence of the antisense strand to form a double-stranded region of about 15 to about 30 base pairs in length. (Item 3) The aforementioned double-stranded region is approximately 17 to 24 base pairs long, as described in item 2 of the RNAi construct. (Item 4) The aforementioned double-stranded region is approximately 19 to 21 base pairs long, and is part of the RNAi construct described in item 2. (Item 5) The RNAi construct described in any one of items 1 to 4, wherein the sense strand and the antisense strand are each independently about 19 to about 30 nucleotides in length. (Item 6) The RNAi construct described in item 5, wherein the sense strand and the antisense strand are each independently about 19 to about 23 nucleotides long. (Item 7) An RNAi construct as described in any one of items 1 to 6, comprising one or two blunt ends. (Item 8) An RNAi construct as described in any one of items 1 to 6, comprising one or two nucleotide overhangs of 1 to 4 unpaired nucleotides. (Item 9) The nucleotide overhang is an RNAi construct as described in item 8, having two unpaired nucleotides. (Item 10) The RNAi construct according to item 8 or 9, comprising a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' ends of both the sense strand and the antisense strand. (Item 11) An RNAi construct described in any one of items 1-10, comprising at least one modified nucleotide. (Item 12) The modified nucleotide is a 2'-modified nucleotide, as described in item 11 of the RNAi construct. (Item 13) The modified nucleotide is a 2'-fluoromodified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a deoxyribonucleotide, or a combination thereof, as described in item 11 of the RNAi construct. (Item 14) The RNAi construct described in item 11, wherein all of the nucleotides in the sense strand and antisense strand are modified nucleotides. (Item 15) The modified nucleotide is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a combination thereof, as described in item 14 of the RNAi construct. (Item 16) The RNAi construct according to any one of items 1 to 15, wherein the sense strand contains a debasalized nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. (Item 17) The RNAi construct described in item 16, wherein the debasalized nucleotide is bound to an adjacent nucleotide via a 3'-3' nucleotide bond or a 5'-5' nucleotide bond. (Item 18) The RNAi construct according to any one of items 1 to 17, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include one or more phosphorothioate nucleotide interlinks. (Item 19) The RNAi construct described in item 18, wherein the antisense strand includes two consecutive phosphorothioate nucleotide interlinks between both the 3' and 5' terminal nucleotides. (Item 20) The RNAi construct described in item 18 or 19, wherein the sense strand includes a single phosphorothioate nucleotide bond between the terminal nucleotides at the 3' end. (Item 21) The RNAi construct according to item 18 or 19, wherein the sense strand includes two consecutive phosphorothioate nucleotide interlinks between the terminal nucleotides at the 3' end. (Item 22) The RNAi construct described in any one of items 1 to 21, wherein the antisense strand includes or consists of a sequence selected from the antisense sequences listed in Table 1 or Table 2. (Item 23) The RNAi construct described in any one of items 1 to 22, wherein the antisense strand includes or consists of a sequence selected from SEQ ID NO: 715, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 738, SEQ ID NO: 754, SEQ ID NO: 761, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 766, SEQ ID NO: 809, SEQ ID NO: 810, SEQ ID NO: 814, SEQ ID NO: 841, SEQ ID NO: 848, SEQ ID NO: 851, SEQ ID NO: 862, SEQ ID NO: 916, SEQ ID NO: 1057, SEQ ID NO: 1078, SEQ ID NO: 2919, SEQ ID NO: 2926, SEQ ID NO: 2946, SEQ ID NO: 2949, SEQ ID NO: 2953 and SEQ ID NO: 2956. (Item 24) The RNAi construct described in any one of items 1 to 23, wherein the sense strand includes or consists of a sequence selected from the sense sequences listed in Table 1 or Table 2. (Item 25) The RNAi construct described in item 24, wherein the sense strand includes or consists of a sequence selected from SEQ ID NO: 46, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 172, SEQ ID NO: 179, SEQ ID NO: 182, SEQ ID NO: 193, SEQ ID NO: 247, SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 409, SEQ ID NO: 2808 and SEQ ID NO: 2820. (Item 26) (i) The sense strand contains or consists of the sequence of sequence number 46, and the an The chicens chain contains or consists of the sequence of sequence number 715. (ii) The sense strand contains or consists of the sequence of sequence number 63, and the antisense strand contains or consists of the sequence of sequence number 732, (iii) The sense strand contains or consists of the sequence of sequence number 64, and the antisense strand contains or consists of the sequence of sequence number 733, (iv) The sense strand contains or consists of the sequence of sequence number 69, and the antisense strand contains or consists of the sequence of sequence number 738, (v) The sense strand contains or consists of the sequence of sequence number 85, and the antisense strand contains or consists of the sequence of sequence number 754, (vi) The sense strand contains or consists of the sequence of sequence number 92, and the antisense strand contains or consists of the sequence of sequence number 761, (vii) The sense strand contains or consists of the sequence of sequence number 94, and the antisense strand contains or consists of the sequence of sequence number 763, (viii) The sense strand contains or consists of the sequence of sequence number 95, and the antisense strand contains or consists of the sequence of sequence number 764, (ix) The sense strand contains or consists of the sequence of sequence number 97, and the antisense strand contains or consists of the sequence of sequence number 766, (x) The sense strand contains or consists of the sequence of sequence number 140, and the antisense strand contains or consists of the sequence of sequence number 809. (xi) The sense strand comprises or consists of the sequence of sequence number 141, and the antisense strand comprises or consists of the sequence of sequence number 810. (xii) The sense strand contains or consists of the sequence of sequence number 145, and the antisense strand contains or consists of the sequence of sequence number 814, (xiii) The sense strand contains or consists of the sequence of sequence number 172, and the antisense strand contains or consists of the sequence of sequence number 841, (xiv) The sense strand contains or consists of the sequence of sequence number 179, and the antisense strand contains or consists of the sequence of sequence number 848, (xv) The sense strand contains or consists of the sequence of sequence number 182, and the antisense strand contains or consists of the sequence of sequence number 851, (xvi) The sense strand contains or consists of the sequence of sequence number 193, and the antisense strand contains or consists of the sequence of sequence number 862, or (xvii) The RNAi construct according to any one of items 1 to 25, wherein the sense strand contains or consists of the sequence of SEQ ID NO: 247, and the antisense strand contains or consists of the sequence of SEQ ID NO: 916. (Item 27) (i) The sense strand contains or consists of the sequence of sequence number 409, and the antisense strand contains or consists of the sequence of sequence number 1078, (ii) The sense strand contains or consists of the sequence of sequence number 388, and the antisense strand contains or consists of the sequence of sequence number 1057, (iii) The sense strand contains or consists of the sequence of sequence number 2808, and the antisense strand contains or consists of the sequence of sequence number 2926, (iv) The sense strand contains or consists of the sequence of sequence number 2820, and the antisense strand contains or consists of the sequence of sequence number 2946, (v) The sense strand contains or consists of the sequence of sequence number 391, and the antisense strand contains or consists of the sequence of sequence number 2949, (vi) The sense strand comprises or consists of the sequence of sequence number 390, and the antisense strand comprises or consists of the sequence of sequence number 2956, (vii) The sense strand contains or consists of the sequence of sequence number 179, and the antisense strand contains or consists of the sequence of sequence number 2919, (viii) The sense strand contains or consists of the sequence of sequence number 388, and the The antisense strand contains or consists of the sequence of sequence number 2953, or (ix) The RNAi construct according to any one of items 1 to 25, wherein the sense strand contains or consists of the sequence of SEQ ID NO: 388, and the antisense strand contains or consists of the sequence of SEQ ID NO: 1057. (Item 28) (i) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3078, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3337. (ii) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3080, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3339. (iii) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3163, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3441. (iv) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3183, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3469 (v) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3076, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3472. (vi) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3077, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3484. (vii) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 2051, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3545. (viii) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3080, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3481. (ix) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3188, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3339. (x) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3080, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3476, or (xi) The RNAi construct according to item 27, wherein the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3223, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3517. (Item 29) An RNAi construct described in any of items 1 to 28, which is one of the double-stranded compounds listed in Tables 1 to 24. (Item 30) The RNAi constructs listed in item 29, which are D-2078, D-2079, D-2081, D-2182, D-2196, D-2238, D-2241, D-2243, D-2246, D-2255, D-2258, D-2301, D-2316, D-2317, D-2329, D-2332, D-2341, D-2344, D-2356, D-2357, D-2399, or D-2510. (Item 31) D-2079, D-2081, D-2196, D-2238, D-2241, D-2255, D-2258, D-2317, D-2332, D-2357 or D-2399 The RNAi construct described in item 30. (Item 32) An RNAi construct for inhibiting the expression of the human MARC1 gene in a cell, comprising a sense strand and an antisense strand that hybridize to form a double-stranded region of about 15 to about 30 base pairs in length, wherein the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1205 to 1250 of SEQ ID NO: 1. (Item 33) The RNAi construct according to item 32, wherein the region of the antisense strand comprises a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1209 to 1239 of SEQ ID NO: 1. (Item 34) The RNAi construct described in item 32 or 33, wherein the region of the antisense strand includes the sequence CAUCUAAUAUUCCAG (SEQ ID NO: 3656). (Item 35) D-2063, D-2066, D-2076, D-2077, D-2078, D-2080, D-2081, D-2108, D-2113, D-2142, D-2240, D-2241, D-2243, D-2245, D-2246, D-2248, D-2250, D-2251, D-2253, D-2255, D-2256, D-2258, D-2259, D-2261, D-2264, D-2265, D-2268, D-2269, D-2270, D-2271, D-2301, D-2309, D-2311, D-2312, D-2314, D-2316, D-2317, D-2319, D-2321, D-2322, D-2324, D-2326, D-2327, D-2329, D-2331, D-2332, D-2334, D-2336, D -2337, D-2339, D-2341, D-2342, D-2344, D-2346, D-2347, D-2349, D-2351, D-2352, D-2354, D-2356, D -2357, D-2376, D-2380, D-2393, D-2395, D-2396, D-2431, D-2436, D-2437, D-2440, D-2441, D-2444, D- The RNAi constructs listed in item 32, which are 2445, D-2447, D-2453, D-2518, D-2519, D-2520, D-2521, D-2522, D-2523, D-2524, D-2525, D-2526, D-2527, D-2528, D-2529, D-2530, D-2531, D-2532, D-2533, D-2534, or D-2535. (Item 36) The RNAi construct described in item 35, which is D-2063, D-2066, D-2076, D-2077, D-2078, D-2080, D-2081, D-2108, D-2113, D-2142, or D-2301. (Item 37) An RNAi construct for inhibiting the expression of the human MARC1 gene in a cell, comprising a sense strand and an antisense strand that hybridize to form a double-stranded region of about 15 to about 30 base pairs in length, wherein the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1345 to 1375 of SEQ ID NO: 1. (Item 38) The region of the antisense strand comprises the RNAi construct described in item 37, wherein the region comprises the sequence UGGGACAUUGAAGCA (SEQ ID NO: 3657). (Item 39) D-2042, D-2043, D-2047, D-2052, D-2158, D-2162, D-2169, D-2182, D-2183, D-2184, D-2185, D- 2186, D-2187, D-2189, D-2211, D-2213, D-2304, D-2305, D-2306, D-2307, D-2308, D-2384, D-238 5, the RNAi construct described in item 37, which is D-2386, D-2387, D-2388, D-2389, D-2390, D-2391, D-2392, D-2399, D-2400, D-2401, D-2402, D-2403, D-2488, D-2494, D-2500, D-2506, D-2512, D-2538, D-2539, D-2540, or D-2541. (Item 40) The RNAi construct described in item 39, which is D-2042, D-2043, D-2047, D-2052, D-2304, D-2305, D-2306, D-2307, or D-2308. (Item 41) An RNAi construct for inhibiting the expression of the human MARC1 gene in a cell, comprising a sense strand and an antisense strand that hybridize to form a double-stranded region of about 15 to about 30 base pairs in length, wherein the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2039 to 2078 of SEQ ID NO: 1. (Item 42) The RNAi construct according to item 41, wherein the region of the antisense strand comprises a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2048 to 2074 of SEQ ID NO: 1. (Item 43) The RNAi construct described in item 41 or 42, wherein the region of the antisense strand comprises the sequence AUCAGAUCUUAGAGU (SEQ ID NO: 3658). (Item 44) D-2045, D-2065, D-2079, D-2082, D-2105, D-2106, D-2137, D-2143, D-2166, D-2173, D-2193, D-2242, D-2247, D-2252, D-2257, D-2260, D-2262, D-2266, D-2272, D-2273, D-2302, D-2303, D-2310, D-2313, D-2315, D-2318, D-2320, D-2323, D-2325, D-2328, D-2330, D-2333, D-2335, D-2338, D-2340, D-2343, D RNAi constructs listed in item 41, which are D-2345, D-2348, D-2350, D-2353, D-2355, D-2358, D-2394, D-2397, D-2454, D-2455, D-2456, D-2457, D-2458, D-2459, D-2460, D-2463, D-2465, D-2468, D-2470, D-2472, D-2473, D-2477, D-2487, D-2493, D-2499, D-2505, D-2511, D-2552, D-2553, D-2554, D-2555, D-2556, or D-2557. (Item 45) The RNAi constructs listed in item 44, which are D-2045, D-2065, D-2079, D-2082, D-2105, D-2106, D-2137, D-2143, D-2302, or D-2303. (Item 46) The aforementioned double-stranded region is an RNAi construct described in any one of items 32 to 45, having a length of approximately 19 to 21 base pairs. (Item 47) The RNAi construct according to any one of items 32 to 46, wherein the sense strand and the antisense strand are each independently about 19 to about 30 nucleotides in length. (Item 48) The RNAi construct described in item 47, wherein the sense strand and the antisense strand are each independently about 19 to about 23 nucleotides long. (Item 49) An RNAi construct as described in any one of items 32-48, comprising one or two blunt ends. (Item 50) An RNAi construct as described in any one of items 32-48, comprising one or two nucleotide overhangs of one to four unpaired nucleotides. (Item 51) The nucleotide overhang is an RNAi construct as described in item 50, having two unpaired nucleotides. (Item 52) The RNAi construct according to item 50 or 51, comprising a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' ends of both the sense strand and the antisense strand. (Item 53) An RNAi construct described in any one of items 32-52, comprising at least one modified nucleotide. (Item 54) The modified nucleotide is a 2'-modified nucleotide, as described in item 53 of the RNAi construct. (Item 55) The modified nucleotide is a 2'-fluoromodified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, BNA, a deoxyribonucleotide, or a combination thereof, as described in item 54 of the RNAi construct. (Item 56) The RNAi construct described in item 53, wherein all of the nucleotides in the sense strand and antisense strand are modified nucleotides. (Item 57) The modified nucleotide is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a combination thereof, as described in item 56 of the RNAi construct. (Item 58) The RNAi construct according to any one of items 32 to 57, wherein the sense strand contains a debasalized nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. (Item 59) The RNAi construct described in item 58, wherein the debasalized nucleotide is bound to an adjacent nucleotide via a 3'-3' nucleotide bond or a 5'-5' nucleotide bond. (Item 60) The RNAi construct according to any one of items 32 to 59, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include one or more phosphorothioate nucleotide interlinks. (Item 61) The RNAi construct described in item 60, wherein the antisense strand includes two consecutive phosphorothioate internucleotide bonds between both the 3' and 5' terminal nucleotides. (Item 62) The RNAi construct according to item 60 or 61, wherein the sense strand includes a single phosphorothioate nucleotide bond between the terminal nucleotides at the 3' end. (Item 63) The RNAi construct according to item 60 or 61, wherein the sense strand includes two consecutive phosphorothioate nucleotide interlinks between the terminal nucleotides at the 3' end. (Item 64) An RNAi construct, further comprising a ligand, as described in any one of items 1 through 63. (Item 65) The ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folic acid moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or its antigen-binding fragment, as described in item 64. (Item 66) The ligand is an RNAi construct as described in item 64, comprising galactose, galactosamine, or N-acetyl-galactosamine. (Item 67) The ligand is an RNAi construct as described in item 66, comprising a polyvalent galactose moiety or a polyvalent N-acetyl-galactosamine moiety. (Item 68) The RNAi construct described in item 67, wherein the polyvalent galactose moiety or polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. (Item 69) The RNAi construct according to any one of items 64 to 68, wherein the ligand is optionally covalently bound to the sense strand via a linker. (Item 70) The ligand is covalently bound to the 5' end of the sense strand in the RNAi construct described in item 69. (Item 71) A pharmaceutical composition comprising an RNAi construct described in any one of items 1 to 70 and a pharmaceutically acceptable carrier or excipient. (Item 72) A method for reducing the expression of the mARC1 protein in a patient in need thereof, comprising administering to the patient an RNAi construct described in any one of items 1 to 70 or a pharmaceutical composition described in item 71. (Item 73) The method according to item 72, wherein the expression level of mARC1 in hepatocytes is reduced in the patient after administration of the RNAi construct or pharmaceutical composition compared to the mARC1 expression level in the patient who has not received the RNAi construct or pharmaceutical composition. (Item 74) The patient is diagnosed with or at risk of cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cirrhosis, as described in item 72. (Item 75) A method for reducing serum cholesterol in a patient in need thereof, comprising administering to the patient an RNAi construct described in any one of items 1 to 70 or a pharmaceutical composition described in item 71. (Item 76) The method according to item 75, wherein the serum cholesterol is non-HDL cholesterol or LDL cholesterol. (Item 77) A method for treating, preventing, or reducing the risk of developing fatty liver disease in a patient in need thereof, comprising administering to the patient an RNAi construct described in any one of items 1 to 70 or a pharmaceutical composition described in item 71. (Item 78) The method according to item 77, wherein the fatty liver disease is non-alcoholic fatty liver disease or non-alcoholic steatohepatitis. (Item 79) The patient is diagnosed with type 2 diabetes, or has a metabolic disorder, or is obese, as described in item 77 or 78. (Item 80) The patient described above has high levels of non-HDL cholesterol or triglycerides, as described in item 77 or 78. (Item 81) A method for treating, preventing or reducing hepatic fibrosis in a patient in need thereof, comprising administering to the patient an RNAi construct described in any one of items 1 to 70 or a pharmaceutical composition described in item 71. (Item 82) The method according to item 81, wherein administering the RNAi construct or pharmaceutical composition to the patient prevents or delays cirrhosis. (Item 83) The method according to item 81 or 82, wherein the patient has been diagnosed with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis. (Item 84) The method according to any one of items 72 to 83, wherein the RNAi construct or pharmaceutical composition is administered to the patient via a parenteral route of administration. (Item 85) The method according to item 84, wherein the parenteral administration route is intravenous or subcutaneous. (Item 86) An RNAi construct described in any one of items 1-70, intended for use in a manner that reduces serum cholesterol in patients in need thereof. (Item 87) The RNAi construct described in item 86, wherein the serum cholesterol is non-HDL cholesterol or LDL cholesterol. (Item 88) An RNAi construct described in any one of items 1 to 70, for use in a manner that treats, prevents, or reduces the risk of developing fatty liver disease in patients in need. (Item 89) The aforementioned fatty liver disease is non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, according to the RNAi construct described in item 88. (Item 90) An RNAi construct described in any one of items 1 to 70, for use in a manner that treats, prevents, or reduces liver fibrosis in patients in need thereof. (Item 91) The patient described above has been diagnosed with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, according to the RNAi construct described in item 90. (Item 92) Use of an RNAi construct described in any one of items 1 to 70 in the preparation of a pharmaceutical product for reducing serum cholesterol in patients in need thereof. (Item 93) The serum cholesterol is non-HDL cholesterol or LDL cholesterol, as described in item 92. (Item 94) Treatment, prevention, or reduction of the risk of developing fatty liver disease in patients who need it. The use of an RNAi construct described in any one of items 1 to 70 in the preparation of a pharmaceutical product for the purpose of [doing so]. (Item 95) The fatty liver disease is non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, as described in item 94. (Item 96) Use of an RNAi construct described in any one of items 1 to 70 in the preparation of a pharmaceutical product for the treatment, prevention, or reduction of hepatic fibrosis in patients in need thereof. (Item 97) The patient has been diagnosed with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, as described in item 96. [Brief explanation of the drawing]

[0016] [Figure 1-1] This shows the nucleotide sequence of the transcript of the human MARC1 gene (Ensembl transcript number ENST00000366910.9; Sequence ID No. 1). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. [Figure 1-2] This shows the nucleotide sequence of the transcript of the human MARC1 gene (Ensembl transcript number ENST00000366910.9; Sequence ID No. 1). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. [Figure 1-3] This shows the nucleotide sequence of the transcript of the human MARC1 gene (Ensembl transcript number ENST00000366910.9; Sequence ID No. 1). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. [Figure 2]These bar graphs show the liver expression of mARC1 mRNA (Figure 2A) and mARC2 mRNA (Figure 2B) in ob / ob mice that were administered subcutaneously with buffer, mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002) every two weeks for six weeks. mRNA levels were evaluated by qPCR at six weeks and are shown compared to mRNA levels in animals that received only buffer injections. [Figure 3-1] This graph shows the serum levels of total cholesterol (CHOL; Figure 3A), LDL cholesterol (LDL; Figure 3B), HDL cholesterol (HDL; Figure 3C), triglycerides (TG; Figure 3D), alanine aminotransferase (ALT; Figure 3E), aspartate aminotransferase (AST; Figure 3F), C-reactive protein (CRP; Figure 3G), and metalloproteinase-1 tissue inhibitor (TIMP-1; Figure 3H) in ob / ob mice that received subcutaneous injections of buffer, mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002) every two weeks for six weeks. Serum levels of various analytes were measured at six weeks using a clinical analytical instrument. Mean ± standard error (SEM) is shown. * = p < 0.05; ** = p < 0.01 vs. buffer control group. [Figure 3-2] This graph shows the serum levels of total cholesterol (CHOL; Figure 3A), LDL cholesterol (LDL; Figure 3B), HDL cholesterol (HDL; Figure 3C), triglycerides (TG; Figure 3D), alanine aminotransferase (ALT; Figure 3E), aspartate aminotransferase (AST; Figure 3F), C-reactive protein (CRP; Figure 3G), and metalloproteinase-1 tissue inhibitor (TIMP-1; Figure 3H) in ob / ob mice that received subcutaneous injections of buffer, mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002) every two weeks for six weeks. Serum levels of various analytes were measured at six weeks using a clinical analytical instrument. Mean ± standard error (SEM) is shown. * = p < 0.05; ** = p < 0.01 vs. buffer control group. [Figure 3-3]This graph shows the serum levels of total cholesterol (CHOL; Figure 3A), LDL cholesterol (LDL; Figure 3B), HDL cholesterol (HDL; Figure 3C), triglycerides (TG; Figure 3D), alanine aminotransferase (ALT; Figure 3E), aspartate aminotransferase (AST; Figure 3F), C-reactive protein (CRP; Figure 3G), and metalloproteinase-1 tissue inhibitor (TIMP-1; Figure 3H) in ob / ob mice that received subcutaneous injections of buffer, mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002) every two weeks for six weeks. Serum levels of various analytes were measured at six weeks using a clinical analytical instrument. Mean ± standard error (SEM) is shown. * = p < 0.05; ** = p < 0.01 vs. buffer control group. [Figure 3-4] This graph shows the serum levels of total cholesterol (CHOL; Figure 3A), LDL cholesterol (LDL; Figure 3B), HDL cholesterol (HDL; Figure 3C), triglycerides (TG; Figure 3D), alanine aminotransferase (ALT; Figure 3E), aspartate aminotransferase (AST; Figure 3F), C-reactive protein (CRP; Figure 3G), and metalloproteinase-1 tissue inhibitor (TIMP-1; Figure 3H) in ob / ob mice that received subcutaneous injections of buffer, mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002) every two weeks for six weeks. Serum levels of various analytes were measured at six weeks using a clinical analytical instrument. Mean ± standard error (SEM) is shown. * = p < 0.05; ** = p < 0.01 vs. buffer control group. [Figure 4] This graph shows the liver levels of triglycerides (liver TG; Figure 4A) or total cholesterol (liver TC; Figure 4B) at 6 weeks in ob / ob mice that received subcutaneous injections of buffer, mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002) every two weeks for 6 weeks. Mean ± SEM values ​​are shown. *** = p < 0.001 vs. buffer control group. [Figure 5]These bar graphs show the liver expression of mARC1 mRNA (Figure 5A) and mARC2 mRNA (Figure 5B) in c57BL / 6 mice fed a standard solid diet (solid diet control) or a 0.2% cholesterol diet (TD190883). Mice fed the 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks either buffer (TD190883 control), mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002). mRNA levels were evaluated by qPCR at 24 weeks and expressed in comparison to mRNA levels in solid diet control animals. [Figure 6-1] This graph shows the serum levels of aspartate aminotransferase (AST; Figure 6A), alanine aminotransferase (ALT; Figure 6B), total cholesterol (Figure 6C), LDL cholesterol (LDL; Figure 6D), HDL cholesterol (HDL; Figure 6E), and triglycerides (Figure 6F) in c57BL / 6 mice fed a standard solid diet (solid diet control) or a 0.2% cholesterol diet (TD190883). Mice fed a 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks with either buffer (TD190883 control), mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002). Serum levels of various analytes were measured at specified time points after administration using a clinical analytical instrument. Mean ± standard error (SEM) is shown. *=p<0.05; **=p<0.01, ***=p<0.001 vs. TD190883 control group. [Figure 6-2]This graph shows the serum levels of aspartate aminotransferase (AST; Figure 6A), alanine aminotransferase (ALT; Figure 6B), total cholesterol (Figure 6C), LDL cholesterol (LDL; Figure 6D), HDL cholesterol (HDL; Figure 6E), and triglycerides (Figure 6F) in c57BL / 6 mice fed a standard solid diet (solid diet control) or a 0.2% cholesterol diet (TD190883). Mice fed a 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks with either buffer (TD190883 control), mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002). Serum levels of various analytes were measured at specified time points after administration using a clinical analytical instrument. Mean ± standard error (SEM) is shown. *=p<0.05; **=p<0.01, ***=p<0.001 vs. TD190883 control group. [Figure 6-3] This graph shows the serum levels of aspartate aminotransferase (AST; Figure 6A), alanine aminotransferase (ALT; Figure 6B), total cholesterol (Figure 6C), LDL cholesterol (LDL; Figure 6D), HDL cholesterol (HDL; Figure 6E), and triglycerides (Figure 6F) in c57BL / 6 mice fed a standard solid diet (solid diet control) or a 0.2% cholesterol diet (TD190883). Mice fed a 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks with either buffer (TD190883 control), mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002). Serum levels of various analytes were measured at specified time points after administration using a clinical analytical instrument. Mean ± standard error (SEM) is shown. *=p<0.05; **=p<0.01, ***=p<0.001 vs. TD190883 control group. [Figure 7-1]This graph shows body weight (Figure 7A), liver weight (Figure 7B), liver triglyceride levels (Figure 7C), and liver total cholesterol levels (Figure 7D) at 24 weeks in c57BL / 6 mice fed a standard solid diet (solid diet control) or a 0.2% cholesterol diet (TD190883). Mice fed the 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks either with buffer (TD190883 control), mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002). Mean ± SEM values ​​are shown. [Figure 7-2] This graph shows body weight (Figure 7A), liver weight (Figure 7B), liver triglyceride levels (Figure 7C), and liver total cholesterol levels (Figure 7D) at 24 weeks in c57BL / 6 mice fed a standard solid diet (solid diet control) or a 0.2% cholesterol diet (TD190883). Mice fed the 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks either with buffer (TD190883 control), mARC1 siRNA (double-stranded number D-1000), or control siRNA (double-stranded number D-1002). Mean ± SEM values ​​are shown. [Figure 8-1] These are the serum concentration-time profiles of the antisense and sense strands of GalNAc-conjugate mARC1 siRNA molecules D-2241 (Figures 8A and 8B), D-2081 (Figures 8C and 8D), and D-2258 (Figures 8E and 8F) after a single 3 mg / kg sc administration in cynomolgus monkeys. Figures 8A, 8C, and 8E show the concentration-time profiles from 0.083 to 24 hours post-administration, while Figures 8B, 8D, and 8F show the concentration-time profiles from 0.083 to 1056 hours post-administration. [Figure 8-2]These are the serum concentration-time profiles of the antisense and sense strands of GalNAc-conjugate mARC1 siRNA molecules D-2241 (Figures 8A and 8B), D-2081 (Figures 8C and 8D), and D-2258 (Figures 8E and 8F) after a single 3 mg / kg sc administration in cynomolgus monkeys. Figures 8A, 8C, and 8E show the concentration-time profiles from 0.083 to 24 hours post-administration, while Figures 8B, 8D, and 8F show the concentration-time profiles from 0.083 to 1056 hours post-administration. [Figure 8-3] These are the serum concentration-time profiles of the antisense and sense strands of GalNAc-conjugate mARC1 siRNA molecules D-2241 (Figures 8A and 8B), D-2081 (Figures 8C and 8D), and D-2258 (Figures 8E and 8F) after a single 3 mg / kg sc administration in cynomolgus monkeys. Figures 8A, 8C, and 8E show the concentration-time profiles from 0.083 to 24 hours post-administration, while Figures 8B, 8D, and 8F show the concentration-time profiles from 0.083 to 1056 hours post-administration. [Modes for carrying out the invention]

[0017] The present invention relates to compositions and methods for regulating the expression of the MARC1 gene in cells or mammals. In some embodiments, the compositions of the present invention include RNAi constructs that target mRNA transcribed from the MARC1 gene, particularly the human MARC1 gene, and reduce the expression of the mARC1 protein in cells or mammals. Such RNAi constructs are useful for reducing serum lipid levels (e.g., total cholesterol and LDL cholesterol levels), treating or preventing various forms of cardiovascular disease and fatty liver diseases such as NAFLD and NASH, and reducing the risk of progression to hepatic fibrosis and cirrhosis.

[0018] As used herein, the term “RNAi construct” refers to a drug comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene (e.g., the MARC1 gene) via an RNA interference mechanism. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other to hybridize to form a double-stranded region. “Hybridizing” typically refers to the pairing of complementary polynucleotides via hydrogen bonds between complementary bases in two polynucleotides (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds). The strand containing a region having a sequence substantially complementary to the target sequence (e.g., target mRNA) is referred to as the “antisense strand” or “guide strand.” The “sense strand” or “passenger strand” refers to a strand containing a region substantially complementary to the antisense strand. In some embodiments, the sense strand may contain a region having a sequence substantially identical to the target sequence.

[0019] Double-stranded RNA molecules may include chemical modifications to ribonucleotides, including modifications to ribose sugars, bases, or ribonucleotide backbone components, such as those described herein or known in the art. Any such modifications used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term “double-stranded RNA” for the purposes of this disclosure.

[0020] As used herein, a polynucleotide containing a first sequence is considered "complementary" to a second sequence if, under certain conditions such as physiological conditions, the first sequence can hybridize to a polynucleotide containing a second sequence to form a double-stranded region. Other such conditions may include moderate or stringent hybridization conditions known to those skilled in the art. A polynucleotide containing a first sequence is considered 100% complementary to a second sequence if it forms base pairs with a polynucleotide containing a second sequence without any mismatches over the entire length of one or both nucleotide sequences. A sequence is considered "substantially complementary" to a target sequence if it is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence. The complementarity percentage can be calculated by dividing the number of bases in the first sequence that are complementary to the bases at the corresponding positions in the second sequence or target sequence by the total length of the first sequence. When two sequences hybridize, if there are 5, 4, 3, or 2 or fewer mismatches across a 30-base-pair double-stranded region, the sequences can also be said to be substantially complementary to the other sequence. In general, if any nucleotide overhangs as defined herein exist, the sequences of such overhangs are not considered in determining the degree of complementarity between the two sequences. As an example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base-pair double-stranded region with a 2-nucleotide overhang at the 3' end of each strand would be considered perfectly complementary when this term is used herein.

[0021] In some embodiments, the antisense strand region includes a sequence substantially or completely complementary to the region of the target RNA sequence (e.g., the mRNA sequence of mARC1). In such embodiments, the sense strand may include a sequence completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may include a sequence substantially complementary to the sequence of the antisense strand, for example, a sequence having 1, 2, 3, 4, or 5 mismatches in the double-stranded region formed by the sense and antisense strands. In certain embodiments, it is preferable that any mismatches occur within the terminal region (e.g., within 6, 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the strand). In one embodiment, any mismatches in the double-stranded region formed by the sense and antisense strands occur within 6, 5, 4, 3, or 2 nucleotides at the 5' end of the antisense strand.

[0022] In certain embodiments, the sense and antisense strands of a double-stranded RNA may hybridize to form a double-stranded region, but otherwise they may be two separate molecules. Such double-stranded RNA molecules formed from two separate strands are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). Therefore, in some embodiments, the RNAi construct of the present invention includes siRNA.

[0023] In other embodiments, the sense and antisense strands that hybridize to form a double-stranded region may be part of a single RNA molecule, i.e., the sense and antisense strands are part of the self-complementary region of the single RNA molecule. In such cases, the single RNA molecule includes a double-stranded region (also referred to as the stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by an adjacent sequence of unpaired nucleotides, thereby forming the loop region. The loop region is typically long enough for the RNA molecule itself to refold so that the antisense strand can base-pair with the sense strand to form a double-stranded or stem region. The loop region can contain about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules having at least partially self-complementary regions are referred to as "small hairpin RNA" (shRNA). In certain embodiments, the RNAi construct of the present invention includes shRNA. The length of a single, at least partially self-complementary RNA molecule may be approximately 40 to 100 nucleotides, approximately 45 to 85 nucleotides, or approximately 50 to 60 nucleotides, and may include double-stranded regions and loop regions having the lengths listed herein, respectively.

[0024] In some embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand, the antisense strand comprising a region having a sequence substantially or completely complementary to the messenger RNA (mRNA) sequence of mARC1. As used herein, “mRNA sequence of mARC1” refers to any messenger RNA sequence including allelic variants and splice variants encoding the mARC1 protein, including variants or isoforms of the mARC1 protein from any species (e.g., non-human primates, humans). The MARC1 gene (also known as MTARC1 or MOSC1) encodes the mitochondrial amidoxime reducing component 1 enzyme (also known as MOCO sulfase C-terminal domain-containing 1 enzyme). In humans, the MARC1 gene is found on chromosome 1 at locus 1q41.

[0025] The mRNA sequence of mARC1 also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. The cDNA sequence refers to the sequence of the mRNA transcript expressed as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Therefore, the antisense strand of the RNAi construct of the present invention may include a region having a sequence substantially or completely complementary to the mRNA sequence or cDNA sequence of the target mARC1. The mRNA or cDNA sequence for mARC1 can be any mRNA or cDNA sequence for mARC1 in the Ensembl genome database or the National Center for Biotechnology Information (NCBI) database, for example: human sequence: Ensembl transcript number ENST00000366910.9 (Figure 1, SEQ ID NO: 1) and NCBI reference sequence NM_022746.4; cynomolgus monkey sequence: NCBI reference sequences XR_001490722.1, XR_001490722.1, XR_001490723.1, XR_001490726.1, XR_273285.2, XM_005540901 Examples include, but are not limited to, XM_005540898.2, XM_005540899.2, rhesus macaque sequences: NCBI reference sequences XM_015115809.2, XM_015115815.2, XM_001102192.4, and XM_001102284.3; chimpanzee sequences: NCBI reference sequences XM_009441519.3, XM_001172926.4, and XM_009441521.3; rat sequences: NCBI reference sequence XM_017598938.1; and mouse sequences: NCBI reference sequence XM_006497192.4. In a particular embodiment, the mRNA sequence of mARC1 is the human transcript shown in Figure 1 (SEQ ID NO: 1).

[0026] The antisense strand region may be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the mRNA sequence of mARC1. In certain embodiments, the antisense strand region includes a sequence substantially complementary to at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotide sequences of a region of the mRNA sequence of mARC1 (e.g., the mRNA sequence of human mARC1 (SEQ ID NO: 1)), with a mismatch of 1, 2, or 3 or fewer. In related embodiments, the antisense strand includes a region having a sequence substantially complementary to at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotide sequences of a region of the mRNA sequence of mARC1, with a mismatch of 1 or fewer. In embodiments where the antisense strand sequence is not perfectly complementary to the target mARC1 mRNA sequence and includes a mismatch, the mismatch may occur between the target mARC1 mRNA sequence and the nucleotides at positions 6 and / or 8 from the 5' end of the antisense strand. In some embodiments, the target region of the mARC1 mRNA sequence in which the antisense strand contains a complementary region may range from about 15 to about 30 consecutive nucleotides, about 16 to about 28 consecutive nucleotides, about 18 to about 26 consecutive nucleotides, about 17 to about 24 consecutive nucleotides, about 19 to about 30 consecutive nucleotides, about 19 to about 25 consecutive nucleotides, about 19 to about 23 consecutive nucleotides, or about 19 to about 21 consecutive nucleotides. In certain embodiments, the region of the antisense strand containing a sequence substantially or perfectly complementary to the mARC1 mRNA sequence may include at least 15 consecutive nucleotides from the antisense sequences listed in Table 1 or Table 2. In other embodiments, the antisense strand sequence includes at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides from the antisense sequences listed in Table 1 or Table 2.

[0027] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to the antisense strand such that the two strands hybridize under physiological conditions to form a double-stranded region. A “double-stranded region” refers to a region of two complementary or substantially complementary polynucleotides that form a double helix between two polynucleotides by base pairing, either through Watson-Crick base pairing or other hydrogen bonding interactions. The double-stranded region of an RNAi construct should be long enough to allow the RNAi construct to enter the RNA interference pathway, for example, by binding to a Dicer enzyme and / or RISC complex. For example, in some embodiments, the double-stranded region is about 15 to about 30 base pairs long. Other lengths of the double-stranded region within this range are also suitable, for example, about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In certain embodiments, the double-stranded region is about 17 to about 24 base pairs long. In other embodiments, the double-stranded region is about 19 to about 21 base pairs long. In one embodiment, the double-stranded region is about 19 base pairs long. In another embodiment, the double-stranded region is about 21 base pairs long.

[0028] In embodiments where the sense and antisense strands are two separate molecules (for example, the RNAi construct contains siRNA), the sense and antisense strands do not need to be the same length as the length of the double-stranded region. For example, one or both strands may be longer than the double-stranded region and have one or more unpaired nucleotides or mismatches adjacent to the double-stranded region. Thus, in some embodiments, the RNAi construct includes at least one nucleotide overhang. As used herein, “nucleotide overhang” refers to an unpaired nucleotide at the end of a strand or a nucleotide that extends beyond the double-stranded region. A nucleotide overhang is typically generated when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of a nucleotide overhang is generally 1–6 nucleotides, 1–5 nucleotides, 1–4 nucleotides, 1–3 nucleotides, 2–6 nucleotides, 2–5 nucleotides, or 2–4 nucleotides. In some embodiments, the nucleotide overhang contains 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang contains 1 to 4 nucleotides. In a particular embodiment, the nucleotide overhang contains 2 nucleotides. In a particular other embodiment, the nucleotide overhang contains a single nucleotide.

[0029] The nucleotides in the overhang may be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides in the overhang are 2'-modified nucleotides (e.g., 2'-fluoromodified nucleotides, 2'-O-methyl modified nucleotides), deoxyribonucleotides, debasalized nucleotides, inverted nucleotides (e.g., inverted debasalized nucleotides, inverted deoxyribonucleotides), or combinations thereof. For example, in one embodiment, the nucleotide in the overhang is a deoxyribonucleotide, e.g., deoxythymidine. In another embodiment, the nucleotide in the overhang is a 2'-O-methyl modified nucleotide, a 2'-fluoromodified nucleotide, a 2'-methoxyethyl modified nucleotide, or combinations thereof. In yet another embodiment, the overhang comprises a 5'-uridine-uridine-3'(5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, e.g., a 2'-modified nucleotide. In other embodiments, the overhang contains a 5'-deoxythymidine-deoxythymidine-3'(5'-dTdT-3') dinucleotide. If the nucleotide overhang is present on the antisense strand, the nucleotides within the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain any other sequence (e.g., polypyrimidine or polypurine sequences, e.g., UU, TT, AA, GG).

[0030] Nucleotide overhangs may be present at the 5' or 3' ends of one or both strands. For example, in one embodiment, the RNAi construct includes nucleotide overhangs at the 5' and 3' ends of the antisense strand. In another embodiment, the RNAi construct includes nucleotide overhangs at the 5' and 3' ends of the sense strand. In some embodiments, the RNAi construct includes nucleotide overhangs at the 5' end of the sense strand and the 5' end of the antisense strand. In yet another embodiment, the RNAi construct includes nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.

[0031] An RNAi construct may contain a single nucleotide overhang at one end of a double-stranded RNA molecule and a blunt end at the other end. “Blunt end” means that the sense and antisense strands are fully base-paired at the ends of the molecule, with no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the RNAi construct contains a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct contains a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct contains blunt ends at both ends of the double-stranded RNA molecule. In these embodiments, the sense and antisense strands are of equal length, and the double-stranded region is of equal length to the sense and antisense strands (i.e., the molecule is double-stranded over its entire length).

[0032] The sense strand and antisense strand in the RNAi construct of the present invention may each independently be about 15 to about 30 nucleotides long, about 19 to about 30 nucleotides long, about 18 to about 28 nucleotides long, about 19 to about 27 nucleotides long, about 19 to about 25 nucleotides long, about 19 to about 23 nucleotides long, about 19 to about 21 nucleotides long, about 21 to about 25 nucleotides long, or about 21 to about 23 nucleotides long. In certain embodiments, the sense strand and antisense strand are each independently about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides long. In some embodiments, the sense strand and antisense strand form a double-stranded region that is the same length but shorter than these strands, so that the RNAi construct has two nucleotide overhangs. For example, in one embodiment, the RNAi construct includes (i) a sense strand and an antisense strand, each 21 nucleotides long, (ii) a 19-base-pair-long double-stranded region, and (iii) nucleotide overhangs of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct includes (i) a sense strand and an antisense strand, each 23 nucleotides long, (ii) a 21-base-pair-long double-stranded region, and (iii) nucleotide overhangs of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In yet another embodiment, the sense strand and antisense strand have the same length and form a double-stranded region along their entire length such that there are no nucleotide overhangs at either end of the double-stranded molecule. In such an embodiment, the RNAi construct is blunt-ended (e.g., has two blunt ends) and includes (i) a sense strand and an antisense strand, each 21 nucleotides long, and (ii) a 21-base-pair-long double-stranded region. In another such embodiment, the RNAi construct is blunt-ended (for example, having two blunt ends) and comprises (i) a sense strand and an antisense strand, each 23 nucleotides long, and (ii) a double-stranded region 23 base pairs long.In yet another such embodiment, the RNAi construct is blunt-ended (for example, having two blunt ends) and comprises (i) a sense strand and an antisense strand, each 19 nucleotides long, and (ii) a double-stranded region 19 base pairs long.

[0033] In other embodiments, the RNAi construct includes at least one nucleotide overhang, such that one sense strand or antisense strand is longer than the other, and the two strands form a double-stranded region having a length equal to the length of the shorter strand. For example, in one embodiment, the RNAi construct includes (i) a 19-nucleotide sense strand, (ii) a 21-nucleotide antisense strand, (iii) a 19-base-pair-length double-stranded region, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct includes (i) a 21-nucleotide sense strand, (ii) a 23-nucleotide antisense strand, (iii) a 21-base-pair-length double-stranded region, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.

[0034] The antisense strand of the RNAi construct of the present invention may include or consist of any one of the antisense sequences listed in Table 1 or Table 2, the sequences of nucleotides 1 to 19 of any of these antisense sequences, or the sequences of nucleotides 2 to 19 of any of these antisense sequences. Accordingly, in some embodiments, the antisense strand includes or consists of a sequence selected from SEQ ID NOs. 671 to 1339, 2072 to 2803, 2906 to 3061, or 3321 to 3655. In other embodiments, the antisense strand includes or consists of the sequences of nucleotides 1 to 19 of any one of SEQ ID NOs. 671 to 1339, 2072 to 2803, 2906 to 3061, or 3321 to 3655. In yet another embodiment, the antisense strand comprises or consists of one sequence of nucleotides 2 to 19 from sequence numbers 671 to 1339, 2072 to 2803, 2906 to 3061, or 3321 to 3655.In a particular embodiment, the antisense chain is sequence numbers 715, 725, 732, 733, 737, 738, 739, 745, 754, 757, 758, 761, 762, 763, 764, 766, 767, 768, 770, 782, 784, 801, 809, 810, 811, 814, 818, 821, 837, 841, 842, 845, 847, 848, It includes or consists of sequences selected from 850, SEQ ID NO: 851, SEQ ID NO: 855, SEQ ID NO: 856, SEQ ID NO: 860, SEQ ID NO: 861, SEQ ID NO: 862, SEQ ID NO: 865, SEQ ID NO: 875, SEQ ID NO: 884, SEQ ID NO: 886, SEQ ID NO: 891, SEQ ID NO: 899, SEQ ID NO: 901, SEQ ID NO: 907, SEQ ID NO: 914, SEQ ID NO: 916, SEQ ID NO: 920, SEQ ID NO: 927, SEQ ID NO: 937, SEQ ID NO: 1056, SEQ ID NO: 1057, SEQ ID NO: 1058, SEQ ID NO: 1059, SEQ ID NO: 1078, SEQ ID NO: 2917, SEQ ID NO: 2919, SEQ ID NO: 2926, SEQ ID NO: 2946, SEQ ID NO: 2949, SEQ ID NO: 2951, SEQ ID NO: 2953, and SEQ ID NO: 2956.In some embodiments, the antisense chain is sequence numbers 715, 732, 733, 737, 738, 739, 745, 754, 757, 761, 762, 763, 764, 766, 767, 784, 801, 809, 810, 811, 814, 841, 842, 845 , containing or consisting of sequences selected from SEQ ID NOs. 848, 851, 856, 860, 862, 914, 916, 927, 937, 1056, 1057, 1058, 1059, 1078, 2917, 2919, 2926, 2946, 2949, 2951, 2953, and 2956. In other embodiments, the antisense chain includes or consists of sequences selected from SEQ ID NOs: 715, 732, 733, 738, 754, 761, 763, 764, 766, 809, 810, 814, 841, 848, 851, 862, 916, 1057, 1078, 2919, 2926, 2946, 2949, 2953, and 2956.

[0035] In these and other embodiments, the sense strand of the RNAi construct of the present invention may include or consist of any one of the sense sequences listed in Table 1 or Table 2, the sequence of nucleotides 1 to 19 of any of these sense sequences, or the sequence of nucleotides 2 to 19 of any of these sense sequences. Accordingly, in some embodiments, the sense strand includes or consists of a sequence selected from SEQ ID NOs: 2 to 670, 1340 to 2071, 2804 to 2905, or 3062 to 3320. In other embodiments, the sense strand includes or consists of the sequence of nucleotides 1 to 19 of any one of SEQ ID NOs: 2 to 670, 1340 to 2071, 2804 to 2905, or 3062 to 3320. In yet another embodiment, the sense strand includes or consists of the sequence of nucleotides 2 to 19 of any one of SEQ ID NOs: 2 to 670, 1340 to 2071, 2804 to 2905, or 3062 to 3320. In a particular embodiment, the sense chain is sequence numbers 46, 56, 63, 64, 68, 69, 70, 76, 85, 88, 89, 92, 93, 94, 95, 97, 98, 99, 101, 113, 115, 132, 140, 141, 142, 145, 149, 152, 168, 172, 173, 176, 178, It contains or consists of sequences selected from sequence number 179, sequence number 181, sequence number 182, sequence number 186, sequence number 187, sequence number 191, sequence number 192, sequence number 193, sequence number 196, sequence number 206, sequence number 215, sequence number 217, sequence number 222, sequence number 230, sequence number 232, sequence number 238, sequence number 245, sequence number 247, sequence number 251, sequence number 258, sequence number 268, sequence number 387, sequence number 388, sequence number 389, sequence number 390, sequence number 391, sequence number 392, sequence number 409, sequence number 2808 and sequence number 2820.In certain other embodiments, the sense chain includes or consists of sequences selected from SEQ ID NOs: 46, 63, 64, 68, 69, 70, 76, 85, 88, 92, 93, 94, 95, 97, 98, 115, 132, 140, 141, 142, 145, 172, 173, 176, 179, 182, 187, 191, 193, 245, 247, 258, 268, 387, 388, 389, 390, 391, 392, 409, 2808, and 2820. In yet another embodiment, the sense chain includes or consists of sequences selected from SEQ ID NOs: 46, 63, 64, 69, 85, 92, 94, 95, 97, 140, 141, 145, 172, 179, 182, 193, 247, 388, 390, 391, 409, 2808, and 2820.

[0036] In certain embodiments of the present invention, the RNAi construct comprises (i) a sense strand comprising or consisting of a sequence selected from 2-670, 1340-2071, 2804-2905 or 3062-3320, and (ii) an antisense strand comprising or consisting of a sequence selected from sequence numbers 671-1339, 2072-2803, 2906-3061 or 3321-3655. In some embodiments, the RNAi construct is (i) SEQ ID NOs. 46, 56, 63, 64, 68, 69, 70, 76, 85, 88, 89, 92, 93, 94, 95, 97, 98, 99, 101, 113, 115, 132, 140, 141, 142, 145, 149, 152, 168, 172, 173, 176, 178, 179, 181, 182, 186, 187, 191, 192, 193, 196, 206, 215, 217, 222, 230, 232, 238, 245, A sense chain containing or consisting of sequences selected from SEQ ID NOs: 247, 251, 258, 268, 387, 388, 389, 390, 391, 392, 409, 2808 and 2820, and (ii) SEQ ID NOs: 715, 725, 732, 733, 737, 738, 739, 745, Number 754, Sequence ID 757, Sequence ID 758, Sequence ID 761, Sequence ID 762, Sequence ID 763, Sequence ID 764, Sequence ID 766, Sequence ID 767, Sequence ID 768, Sequence ID 770, Sequence ID 782, Sequence ID 784, Sequence ID 801, Sequence ID 809, Sequence ID 810, Sequence ID 811, Sequence ID 814, Sequence ID 818, Sequence ID 821, Sequence ID 837, Sequence ID 841, Sequence ID 842, Sequence ID 845, Sequence ID 847, Sequence ID 848,Includes an antisense chain containing or consisting of sequences selected from SEQ ID NOs. 850, 851, 855, 856, 860, 861, 862, 865, 875, 884, 886, 891, 899, 901, 907, 914, 916, 920, 927, 937, 1056, 1057, 1058, 1059, 1078, 2917, 2919, 2926, 2946, 2949, 2951, 2953, and 2956. In other embodiments, the RNAi construct includes (i) SEQ ID NOs: 46, 63, 64, 68, 69, 70, 76, 85, 88, 92, 93, 94, 95, 97, 98, 115, 132, 140, 141, 142, 145, 172, 173, 176, 179, 182, 187, 191, 193, 245, 247, 258, 268, 387, 388, 389, 390, 391, 392, 409, 2808 and SEQ ID NOs: 2 A sense chain containing or consisting of sequences selected from 820, and (ii) SEQ ID NOs: 715, 732, 733, 737, 738, 739, 745, 754, 757, 761, 762, 763, 764, 766, 767, 784, 801, 809, 810, 811, 814, 841, 842, 845, 848, 851, 856, 860, 862, 914, 916, 927, 937, 1056, 1057, 1058, 1059, 1078,In further embodiments, the RNAi construct includes (i) a sense strand containing or consisting of a sequence selected from SEQ ID NOs: 2917, 2919, 2926, 2946, 2949, 2951, 2953 and 2956. Includes an antisense chain containing or consisting of sequences selected from sequence number 715, sequence number 732, sequence number 733, sequence number 738, sequence number 754, sequence number 761, sequence number 763, sequence number 764, sequence number 766, sequence number 809, sequence number 810, sequence number 814, sequence number 841, sequence number 848, sequence number 851, sequence number 862, sequence number 916, sequence number 1057, sequence number 1078, sequence number 2919, sequence number 2926, sequence number 2946, sequence number 2949, sequence number 2953 and sequence number 2956.

[0037] In certain embodiments, the RNAi construct of the present invention includes (i) a sense strand containing or comprising the sequence of SEQ ID NO: 46 and an antisense strand containing or comprising the sequence of SEQ ID NO: 715; (ii) a sense strand containing or comprising the sequence of SEQ ID NO: 63 and an antisense strand containing or comprising the sequence of SEQ ID NO: 732; (iii) a sense strand containing or comprising the sequence of SEQ ID NO: 64 and an antisense strand containing or comprising the sequence of SEQ ID NO: 733; (iv) a sense strand containing or comprising the sequence of SEQ ID NO: 69 and an antisense strand containing or comprising the sequence of SEQ ID NO: 738; (v) a sense strand containing or comprising the sequence of SEQ ID NO: 85 and an antisense strand containing or comprising the sequence of SEQ ID NO: 754; (vi) a sense strand containing or comprising the sequence of SEQ ID NO: 92 and an antisense strand containing or comprising the sequence of SEQ ID NO: 761; (vii) a sense strand containing or comprising the sequence of SEQ ID NO: 94 and an antisense strand containing or comprising the sequence of SEQ ID NO: 763. (viii) a sense strand containing or consisting of the sequence of sequence number 95 and an antisense strand containing or consisting of the sequence of sequence number 764, (ix) a sense strand containing or consisting of the sequence of sequence number 97 and an antisense strand containing or consisting of the sequence of sequence number 766, (x) a sense strand containing or consisting of the sequence of sequence number 140 and an antisense strand containing or consisting of the sequence of sequence number 809, (xi) a sense strand containing or consisting of the sequence of sequence number 141 and an antisense strand containing or consisting of the sequence of sequence number 810, (xii) a sense strand containing or consisting of the sequence of sequence number 145 and an antisense strand containing or consisting of the sequence of sequence number 814, (xiii) a sense strand containing or consisting of the sequence of sequence number 172 and an antisense strand containing or consisting of the sequence of sequence number 841, (xiv) a sense strand containing or consisting of the sequence of sequence number 179 and an antisense strand containing or consisting of the sequence of sequence number 848,(xv) A sense strand containing or consisting of the sequence of sequence number 182 and an antisense strand containing or consisting of the sequence of sequence number 851; (xvi) A sense strand containing or consisting of the sequence of sequence number 193 and an antisense strand containing or consisting of the sequence of sequence number 862; or (xvii) A sense strand containing or consisting of the sequence of sequence number 247 and an antisense strand containing or consisting of the sequence of sequence number 916.

[0038] In certain other embodiments, the RNAi construct of the present invention includes (i) a sense strand containing or comprising the sequence of SEQ ID NO: 409 and an antisense strand containing or comprising the sequence of SEQ ID NO: 1078, (ii) a sense strand containing or comprising the sequence of SEQ ID NO: 388 and an antisense strand containing or comprising the sequence of SEQ ID NO: 1057, (iii) a sense strand containing or comprising the sequence of SEQ ID NO: 2808 and an antisense strand containing or comprising the sequence of SEQ ID NO: 2926, (iv) a sense strand containing or comprising the sequence of SEQ ID NO: 2820 and an antisense strand containing or comprising the sequence of SEQ ID NO: 2946, (v) a sense strand containing or comprising the sequence of SEQ ID NO: 391 or (vi) a sense strand comprising the sequence of sequence number 2949 or an antisense strand comprising the sequence of sequence number 390 or an antisense strand comprising the sequence of sequence number 2956 or an antisense strand comprising the sequence of sequence number 2956 or an antisense strand comprising the sequence of sequence number 179 or an antisense strand comprising the sequence of sequence number 2919 or an antisense strand comprising the sequence of sequence number 388 or an antisense strand comprising the sequence of sequence number 2953 or an antisense strand comprising the sequence of sequence number 388 or an antisense strand comprising the sequence of sequence number 1057.

[0039] In some embodiments, the RNAi construct of the present invention includes (i) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2009 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2741; (ii) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2011 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2743; (iii) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2012 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2744; (iv) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2013 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2745; (v) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2020 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2752. (vi) an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2035 or a sense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2767 or an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2767 or an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2037 or a sense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2769 or an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2041 or a sense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2773 or an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2042 or a sense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2774 or an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2043 or a sense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2775 or an antisense strand comprising the sequence of modified nucleotides according to SEQ ID NO: 2775,(xi) a sense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2044 and an antisense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2776, (xii) a sense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2045 and an antisense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2777, (xiii) a sense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2051 and an antisense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2783, (xiv) a sense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 2053 and (xv) an antisense strand containing or consisting of a modified nucleotide sequence according to sequence number 2785, a sense strand containing or consisting of a modified nucleotide sequence according to sequence number 2054 and an antisense strand containing or consisting of a modified nucleotide sequence according to sequence number 2786, (xvi) a sense strand containing or consisting of a modified nucleotide sequence according to sequence number 2055 and an antisense strand containing or consisting of a modified nucleotide sequence according to sequence number 2787, or (xvii) a sense strand containing or consisting of a modified nucleotide sequence according to sequence number 2059 and an antisense strand containing or consisting of a modified nucleotide sequence according to sequence number 2791.

[0040] In other embodiments, the RNAi construct of the present invention includes (i) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3078 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3337; (ii) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3080 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3339; (iii) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3163 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3441; (iv) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3183 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3469; (v) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3076 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3472. (vi) an antisense strand comprising (vi) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3077 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3484, (vii) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 2051 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3545, (viii) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3080 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3481, (ix) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3188 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3339, (x) a sense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3080 and an antisense strand containing or comprising a sequence of modified nucleotides according to SEQ ID NO: 3476,Or (xi) a sense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 3223 and an antisense strand containing or consisting of a sequence of modified nucleotides according to SEQ ID NO: 3517.

[0041] The RNAi construct of the present invention may be any of the double-stranded compounds listed in Tables 1 to 24 (including the unmodified and / or modified nucleotide sequences of the compounds). In some embodiments, the RNAi construct is any of the double-stranded compounds listed in Table 1. In other embodiments, the RNAi construct is any of the double-stranded compounds listed in Table 2 (including the unmodified and / or modified nucleotide sequences of the compounds). For a specific purpose, the RNAi construct is D-1044, D-1061, D-1062, D-1067, D-1083, D-1090, D-1092, D-1093, D-1095, D-1138, D-1139, D-1143, D-1170, D-1177, D-1180, D-1191, D-1245, D-2000, D-2002, D-2003, D-2004, D-2011, D-2026, D-2028, D-2032, D-2033, D-20 34, D-2035, D-2036, D-2042, D-2044, D-2045, D-2046, D-2050, D-2078, D-2079, D-2081, D-2182, D-2196, D-2238, D-2241, D-22 43, D-2246, D-2255, D-2258, D-2301, D-2316, D-2317, D-2329, D-2332, D-2341, D-2344, D-2356, D-2357, D-2399 or D-2510. In certain other embodiments, the RNAi construct is D-2079, D-2081, D-2196, D-2238, D-2241, D-2255, D-2258, D-2317, D-2332, D-2357, or D-2399.

[0042] In certain embodiments, the RNAi constructs of the present invention may target specific regions of the human mARC1 transcript sequence. As described in Example 4 and summarized in Table 23, certain RNAi constructs comprising an antisense strand designed to have a sequence complementary to a specific region of the human mARC1 transcript (SEQ ID NO: 1) exhibited superior in vivo knockdown activity of human mARC1 mRNA compared to RNAi constructs comprising an antisense strand complementary to other regions of the transcript. Therefore, in some embodiments of the present invention, RNAi constructs particularly suitable for inhibiting the expression of the human MARC1 gene in cells comprise a sense strand and an antisense strand that hybridize to form a double-stranded region of about 15 to about 30 base pairs in length, where the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1205 to 1250 of SEQ ID NO: 1. In one embodiment, the antisense strand includes a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1209 to 1239 of SEQ ID NO: 1. In another embodiment, the antisense strand includes a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1211 to 1236 of SEQ ID NO: 1. In some such embodiments, the antisense strand has a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1205 to 1250, nucleotides 1209 to 1239, or nucleotides 1211 to 1236 of SEQ ID NO: 1, with one, two, or three or fewer mismatches. In other embodiments, the antisense strand has a sequence that is perfectly complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1205 to 1250, nucleotides 1209 to 1239, or nucleotides 1211 to 1236 of SEQ ID NO: 1.RNAi constructs that target nucleotides 1205-1250 of the human mARC1 transcript include D-2063, D-2066, D-2076, D-2077, D-2078, D-2080, D-2081, D-2108, D-2113, D-2142, D-2240, D-2241, D-2243, D-2245, D-2246, D-2248, D-2250, D-2251, and D-22 53, D-2255, D-2256, D-2258, D-2259, D-2261, D-2264, D-2265, D-2268, D-2269, D-2270, D-2271, D-2301, D-2 309, D-2311, D-2312, D-2314, D-2316, D-2317, D-2319, D-2321, D-2322, D-2324, D-2326, D-2327, D-2329, D- 2331, D-2332, D-2334, D-2336, D-2337, D-2339, D-2341, D-2342, D-2344, D-2346, D-2347, D-2349, D-2351, D -2352, D-2354, D-2356, D-2357, D-2376, D-2380, D-2393, D-2395, D-2396, D-2431, D-2436, D-2437, D-2440, Examples include, but are not limited to, D-2441, D-2444, D-2445, D-2447, D-2453, D-2518, D-2519, D-2520, D-2521, D-2522, D-2523, D-2524, D-2525, D-2526, D-2527, D-2528, D-2529, D-2530, D-2531, D-2532, D-2533, D-2534 and D-2535. In some embodiments, RNAi constructs targeting nucleotides 1205–1250 of the human mARC1 transcript are D-2063, D-2066, D-2076, D-2077, D-2078, D-2080, D-2081, D-2108, D-2113, D-2142, or D-2301. In certain embodiments, RNAi constructs targeting nucleotides 1205–1250, particularly nucleotides 1211–1236, of SEQ ID NO: 1 include an antisense strand containing the sequence 5-CAUCUAAUAUUCCAG-3' (SEQ ID NO: 3656).

[0043] In other embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand that hybridize to form a double-stranded region of about 15 to about 30 base pairs in length, wherein the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1345 to 1375 of SEQ ID NO: 1. In one embodiment, the antisense strand comprises a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1345 to 1375 of SEQ ID NO: 1, with one, two, or three or fewer mismatches. In another embodiment, the antisense strand comprises a sequence that is perfectly complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 1345 to 1375 of SEQ ID NO: 1. Exemplary RNAi constructs that target nucleotides 1345-1375 of the human mARC1 transcript include D-2042, D-2043, D-2047, D-2052, D-2158, D-2162, D-2169, D-2182, D-2183, D-2184, D-2185, D-2186, D-2187, D-2189, D-2211, D-2213, D-2304, D-2305, D-2306, D-23 Examples include, but are not limited to, 07, D-2308, D-2384, D-2385, D-2386, D-2387, D-2388, D-2389, D-2390, D-2391, D-2392, D-2399, D-2400, D-2401, D-2402, D-2403, D-2488, D-2494, D-2500, D-2506, D-2512, D-2538, D-2539, D-2540, and D-2541. In some embodiments, RNAi constructs targeting nucleotides 1345–1375 of the human mARC1 transcript are D-2042, D-2043, D-2047, D-2052, D-2304, D-2305, D-2306, D-2307, or D-2308. In certain embodiments, RNAi constructs targeting nucleotides 1345–1375, particularly nucleotides 1350–1375, of SEQ ID NO: 1 include an antisense strand containing the sequence 5-UGGGACAUUGAAGCA-3' (SEQ ID NO: 3657).

[0044] In further embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand that hybridize to form a double-stranded region of about 15 to about 30 base pairs in length, wherein the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2039 to 2078 of SEQ ID NO: 1. In one embodiment, the antisense strand comprises a region having a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2048 to 2074 of SEQ ID NO: 1. In some such embodiments, the antisense strand has a sequence substantially complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2039 to 2078 or nucleotides 2048 to 2074 of SEQ ID NO: 1, with one, two, or three or fewer mismatches. In other embodiments, the antisense strand has a sequence that is perfectly complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 2039 to 2078 or nucleotides 2048 to 2074 of SEQ ID NO: 1. RNAi constructs that target nucleotides 2039-2078 of the human mARC1 transcript include D-2045, D-2065, D-2079, D-2082, D-2105, D-2106, D-2137, D-2143, D-2166, D-2173, D-2193, D-2242, and D-224. 7, D-2252, D-2257, D-2260, D-2262, D-2266, D-2272, D-2273, D-2302, D-2303, D-23 10, D-2313, D-2315, D-2318, D-2320, D-2323, D-2325, D-2328, D-2330, D-2333, D-2 335, D-2338, D-2340, D-2343, D-2345, D-2348, D-2350, D-2353, D-2355, D-2358, D -2394, D-2397, D-2454, D-2455, D-2456, D-2457, D-2458, D-2459, D-2460, D-2463, Examples include, but are not limited to, D-2465, D-2468, D-2470, D-2472, D-2473, D-2477, D-2487, D-2493, D-2499, D-2505, D-2511, D-2552, D-2553, D-2554, D-2555, D-2556, and D-2557.In certain embodiments, RNAi constructs targeting nucleotides 2039–2078 of the human mARC1 transcript are D-2045, D-2065, D-2079, D-2082, D-2105, D-2106, D-2137, D-2143, D-2302, or D-2303. In certain other embodiments, RNAi constructs targeting nucleotides 2039–2078, particularly nucleotides 2048–2074, of SEQ ID NO: 1 include an antisense strand containing the sequence 5-AUCAGAUCUUAGAGU-3' (SEQ ID NO: 3658).

[0045] The RNAi construct of the present invention may include one or more modified nucleotides. “Modified nucleotide” refers to a nucleotide having one or more chemical modifications to a nucleoside, nucleic acid base, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate. However, RNAi constructs may include combinations of modified nucleotides and ribonucleotides. Incorporation of modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example, by reducing the molecule's sensitivity to nucleases and other degradation processes. Incorporation of modified nucleotides can also enhance the efficacy of the RNAi construct in reducing the expression of a target gene.

[0046] In certain embodiments, modified nucleotides have ribose sugar modifications. These sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring, as well as bicyclic sugar modifications. 2'-Modified nucleotides refer to nucleotides having a pentose ring with substituents other than OH at the 2' position. Such 2' modifications include 2'-H (e.g., deoxyribonucleotides) and 2'-O-alkyl (e.g., -O-C1~C) 10 or -O-C1~C 10Examples of modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (substituted alkyl), 2'-O-allyl (-O-CH2CH=CH2), 2'-C-allyl, 2'-deoxy-2'-fluoro (also called 2'-F or 2'-fluoro), 2'-O-methyl (-OCH3), 2'-O-methoxyethyl (-O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine, and 2'-azide. Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S configuration), 5'-vinyl, and 5'-methoxy.

[0047] "Bicyclic sugar modification" refers to the modification of a pentose ring, in which case the bridge connects two atoms of the ring to form a second ring resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification includes a bridge between the 4' and 2' carbon atoms of the pentose ring. A nucleotide containing a sugar moiety having a bicyclic sugar modification is referred to herein as bicyclic nucleic acid or BNA. Exemplary bicyclic sugar modifications include α-L-methyleneoxy(4'-CH2-O-2') bicyclic nucleic acid (BNA), β-D-methyleneoxy(4'-CH2-O-2')BNA (also referred to as loc nucleic acid or LNA), ethyleneoxy(4'-(CH2)2-O-2')BNA, and aminooxy(4'-CH2-ON(R)-2'(wherein R is H, C1-C) 12 BNA, oxyamino(4'-CH2-N(R)-O-2'(wherein R is H, C1~C) which is an alkyl or protecting group. 12 Alkyl or protecting group))BNA, methyl(methyleneoxy)(4'-CH(CH3)-O-2')BNA (also called restrained ethyl or cEt), methylene-thio(4'-CH2-S-2')BNA, methylene-amino(4'-CH2-N(R)-2')(wherein R is H, C1~C 12Examples of alkyl or protecting groups include (4'-(CH2)3-2')BNA, methyl carbon ring (4'-(CH2)3-2')BNA, and methoxy(ethyleneoxy)(4'-CH(CH2OMe)-O-2')BNA (also known as restricted MOE or cMOE), but are not limited to these. These and other glycosylated nucleotides that can be incorporated into the RNAi construct of the present invention are described in U.S. Patent No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference in their entirety.

[0048] In some embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), deoxyribonucleotides, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, or combinations thereof. In a particular embodiment, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof.

[0049] Both the sense and antisense strands of an RNAi construct may contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains one, two, three, four, five, six, seven, eight, nine, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains one, two, three, four, five, six, seven, eight, nine, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, or a combination thereof.

[0050] In certain embodiments, the modified nucleotides incorporated into one or both strands of the RNAi construct of the present invention have modifications to nucleic acid bases (also referred to herein as “bases”). “Modified nucleic acid bases” or “modified bases” refers to bases other than the naturally occurring purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases can be synthetic or naturally occurring, and include universal bases, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and 6-azou Examples include, but are not limited to, uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0051] In some embodiments, the modified base is a universal base. A “universal base” refers to a base analog that indiscriminately forms base pairs with all of the native bases of RNA and DNA without altering the double helix structure of the resulting double-stranded region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole.

[0052] Other suitable modified bases that can be incorporated into the RNAi construct of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol.10:297-310, 2000 and Peacock et al., J.Org.Chem., Vol.76:7295-7300, 2011, both of which are incorporated herein by reference in their entirety. Those skilled in the art will be well aware that guanine, cytosine, adenine, thymine, and uracil can be substituted with other nucleic acid bases, such as the modified nucleic acid bases described above, without substantially altering the base-pair properties of polynucleotides containing nucleotides having such substituted nucleic acid bases.

[0053] In some embodiments, the sense and antisense strands of an RNAi construct may contain one or more debasalized nucleotides. A “debasalized nucleotide” or “debasalized nucleoside” is a nucleotide or nucleoside that lacks a nucleic acid base at the 1' position of a ribose sugar. In certain embodiments, the debasalized nucleotide is incorporated into the ends of the sense and / or antisense strands of the RNAi construct. In one embodiment, the sense strand contains a debasalized nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In another embodiment, the antisense strand contains a debasalized nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends. In such embodiments where the debasalized nucleotide is a terminal nucleotide, the terminal nucleotide may be an inverted nucleotide, i.e., it may be linked to an adjacent nucleotide via a 3'-3' nucleotide bond (if it is on the 3' end of the strand) or a 5'-5' nucleotide bond (if it is on the 5' end of the strand), rather than a natural 3'-5' nucleotide bond. Debasic nucleotides may also include sugar modifications such as any of the sugar modifications described above. In certain embodiments, debasic nucleotides include 2'-modifications, such as 2'-fluoromodifications, 2'-O-methyl modifications, or 2'-H (deoxy) modifications. In one embodiment, the debasic nucleotide includes a 2'-O-methyl modification. In another embodiment, the debasic nucleotide includes a 2'-H modification (i.e., a deoxy-debasic nucleotide).

[0054] In certain embodiments, the RNAi construct of the present invention may include modified nucleotides incorporated into the sense and antisense strands in a specific pattern, such as the pattern described in International Publication No. 2020 / 123410, which is incorporated in its entirety herein by reference. RNAi constructs having such chemical modification patterns have been shown to have improved gene silencing activity in vivo. In one embodiment, the RNAi construct of the present invention includes a sense strand and an antisense strand containing sequences sufficiently complementary to each other to form a double-stranded region of at least 15 base pairs, where, The nucleotides at positions 2, 7, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides. The nucleotides in the sense strand at positions 8-11 and 13 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides. Neither the sense strand nor the antisense strand contains more than 7 total 2'-fluoromodified nucleotides.

[0055] In other embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand containing sequences sufficiently complementary to each other to form a double-stranded region of at least 19 base pairs, where, The nucleotides at positions 2, 7, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides, the nucleotides at positions 4, 6, 10, and 12 (counting from the 5' end) are optionally 2'-fluoromodified nucleotides, and all other nucleotides in the antisense strand are modified nucleotides other than 2'-fluoromodified nucleotides. The nucleotides in the sense strand that are paired with positions 8-11 and 13 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides, the nucleotides in the sense strand that are paired with positions 3 and 5 (counting from the 5' end) in the antisense strand are, by arbitrary selection, 2'-fluoromodified nucleotides, and all other nucleotides in the sense strand are modified nucleotides other than 2'-fluoromodified nucleotides.

[0056] In these embodiments, modified nucleotides other than 2'-fluoromodified nucleotides may be selected from 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNA, and deoxyribonucleotides. In these and other embodiments, the terminal nucleotides at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand may be debasalized nucleotides or deoxyribonucleotides. In these embodiments, the debasalized nucleotides or deoxyribonucleotides may be inverted (i.e., they are attached to adjacent nucleotides via a 3'-3' nucleotide bond (if on the 3' end of the strand) or a 5'-5' nucleotide bond (if on the 5' end of the strand) rather than a natural 3'-5' nucleotide bond).

[0057] In any of the embodiments described above, the nucleotides at positions 2, 7, 12, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides. In other embodiments, the nucleotides at positions 2, 4, 7, 12, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides. In yet another embodiment, the nucleotides at positions 2, 4, 6, 7, 12, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides. In yet another embodiment, the nucleotides at positions 2, 4, 6, 7, 10, 12, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides. In an alternative embodiment, the nucleotides at positions 2, 7, 10, 12, and 14 (counting from the 5' end) of the antisense strand are 2'-fluoromodified nucleotides. In certain other embodiments, the nucleotides at positions 2, 4, 7, 10, 12, and 14 (counting from the 5' end) in the antisense strand are 2'-fluoromodified nucleotides.

[0058] In any of the above embodiments, the nucleotides in the sense strand at positions paired with positions 3, 8 to 11, and 13 (counting from the 5'-end) in the antisense strand are 2'-fluoro modified nucleotides. In some embodiments, the nucleotides in the sense strand at positions paired with positions 5, 8 to 11, and 13 (counting from the 5'-end) in the antisense strand are 2'-fluoro modified nucleotides. In other embodiments, the nucleotides in the sense strand at positions paired with positions 3, 5, 8 to 11, and 13 (counting from the 5'-end) in the antisense strand are 2'-fluoro modified nucleotides.

[0059] In some embodiments, the RNAi construct of the present invention comprises a structure represented by formula (A).

Chemical formula

[0060] In formula (A), the upper strand listed in the 5' to 3' direction is the sense strand, the lower strand listed in the 3' to 5' direction is the antisense strand, and each N F represents a 2'-fluoro modified nucleotide, and each N M independently represents a modified nucleotide selected from 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and each N L independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, and N Tx represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N A x can be an integer from 0 to 4, provided that one or more nucleotides are independently modified nucleotides selected from debasalized nucleotides, inverted debasalized nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. A One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y can be an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N B z can be an integer from 0 to 4, provided that one or more nucleotides are independently modified nucleotides selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. B One or more nucleotides are present in the sense strand when N A They may be complementary to nucleotides, or they may be overhanging nucleotides that do not base-pair with nucleotides in the sense strand.

[0061] In some embodiments of the RNAi construct, which includes the structure represented by formula (A), there is a nucleotide overhang at the 3' end of the sense strand (i.e., y is 1, 2, 3, or 4). In one such embodiment, y is 2. In embodiments where there is a 2-nucleotide overhang at the 3' end of the sense strand (i.e., y is 2), x is 0 and z is 2, or x is 1 and z is 2. In other embodiments of the RNAi construct, which includes the structure represented by formula (A), the RNAi construct has blunt ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In these embodiments where there is no nucleotide overhang at the 3' end of the sense strand (i.e., y is 0), (i) x is 2 and z is 4, (ii) x is 3 and z is 4, (iii) x is 0 and z is 2, (iv) x is 1 and z is 2, or (v) x is 2 and z is 2. In any embodiment where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand is N A The nucleotide may be an inverted nucleotide, such as an inverted debasalized nucleotide or an inverted deoxyribonucleotide.

[0062] In certain embodiments, the RNAi construct includes a structure represented by formula (A), in which the N at positions 4 and 12, counting from the 5' end of the antisense strand, is present. M These are 2'-fluoromodified nucleotides, respectively. In other embodiments, the N at positions 4, 6, and 12 counting from the 5' end of the antisense strand are M These are 2'-fluoromodified nucleotides, respectively. In yet another embodiment, the N3N3 at positions 4, 6, 10, and 12, counting from the 5' end of the antisense strand. M These are 2'-fluoromodified nucleotides, respectively. In alternative embodiments in which the RNAi construct includes the structure represented by formula (A), the N10 and N12 positions, counting from the 5' end of the antisense strand, are present. MThese are 2'-fluoromodified nucleotides, respectively. In related embodiments, the N at positions 4, 10, and 12 counting from the 5' end of the antisense strand. M These are 2'-fluoromodified nucleotides, respectively. In other alternative embodiments in which the RNAi construct includes the structure represented by formula (A), the N at positions 4, 6 and 10, counting from the 5' end of the antisense strand, are present. M These are 2'-O-methyl modified nucleotides, and are located at the 12th position from the 5' end of the antisense strand. M is a 2'-fluoromodified nucleotide. In some embodiments, the RNAi construct includes a structure represented by formula (A), in which each N in the sense strand M is a 2'-O-methyl modified nucleotide. In other embodiments, each N in the sense strand M is a 2'-fluoromodified nucleotide. In yet another embodiment in which the RNAi construct includes a structure represented by formula (A), each N is present in both the sense strand and the antisense strand. M This is a 2'-O-methyl modified nucleotide.

[0063] In any of the above embodiments, the RNAi construct includes a structure represented by formula (A), in which each N in both the sense strand and the antisense strand. L This can be a 2'-O-methyl modified nucleotide. In these embodiments and in any of the embodiments described above, N in formula (A) T This may be an inverted debasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0064] In another embodiment of the present invention, the RNAi construct of the present invention includes a structure represented by formula (B). [ka]

[0065] In equation (B), the upper chains, which are listed in the direction from 5' to 3', are sense chains, and the lower chains, which are listed in the direction from 3' to 5', are antisense chains, and each N F represents a 2'-fluoromodified nucleotide, and each N M Each N independently represents a modified nucleotide selected from 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, BNA, and deoxyribonucleotides. L This independently represents a modified nucleotide selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides, N T x represents a modified nucleotide selected from debasic nucleotides, inverted debasic nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. When x is 1, 2, 3, or 4, N A x can be an integer from 0 to 4, provided that one or more nucleotides are independently modified nucleotides selected from debasalized nucleotides, inverted debasalized nucleotides, inverted deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. A One or more nucleotides may be complementary to the nucleotides in the antisense strand. When y is 1, 2, 3, or 4, y can be an integer from 0 to 4, provided that one or more n nucleotides are modified or unmodified overhang nucleotides that do not base-pair with the nucleotides in the antisense strand. When z is 1, 2, 3, or 4, N Bz can be an integer from 0 to 4, provided that one or more nucleotides are independently modified nucleotides selected from 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, BNA, and deoxyribonucleotides. B One or more nucleotides are present in the sense strand when N A They may be complementary to nucleotides, or they may be overhanging nucleotides that do not base-pair with nucleotides in the sense strand.

[0066] In some embodiments of the RNAi construct, which includes the structure represented by formula (B), there is a nucleotide overhang at the 3' end of the sense strand (i.e., y is 1, 2, 3, or 4). In one such embodiment, y is 2. In embodiments where there is a 2-nucleotide overhang at the 3' end of the sense strand (i.e., y is 2), x is 0 and z is 2, or x is 1 and z is 2. In other embodiments of the RNAi construct, which includes the structure represented by formula (B), the RNAi construct has blunt ends at the 3' end of the sense strand and the 5' end of the antisense strand (i.e., y is 0). In these embodiments where there is no nucleotide overhang at the 3' end of the sense strand (i.e., y is 0), (i) x is 2 and z is 4, (ii) x is 3 and z is 4, (iii) x is 0 and z is 2, (iv) x is 1 and z is 2, or (v) x is 2 and z is 2. In any embodiment where x is greater than 0, the terminal nucleotide at the 5' end of the sense strand is N A The nucleotide may be an inverted nucleotide, such as an inverted debasalized nucleotide or an inverted deoxyribonucleotide.

[0067] In certain embodiments, the RNAi construct includes a structure represented by formula (B), where the N13 is located at positions 4, 6, 8, 9, and 16 counting from the 5' end of the antisense strand. MThese are 2'-fluoromodified nucleotides, and are located at positions 7 and 12 of the antisense strand, counting from the 5' end. M These are 2'-O-methyl-modified nucleotides, respectively. In other embodiments, the N at positions 4 and 6, counting from the 5' end of the antisense strand. M These are 2'-fluoromodified nucleotides, and are located at positions 7-9 from the 5' end of the antisense strand. M These are 2'-O-methyl-modified nucleotides, respectively. In yet another embodiment, the N3s at positions 4, 6, 8, 9 and 16, counting from the 5' end of the antisense strand, are also present. M These are 2'-O-methyl modified nucleotides, and are located at positions 7 and 12 of the antisense strand, counting from the 5' end. M These are 2'-fluoromodified nucleotides, respectively. In alternative embodiments in which the RNAi construct includes the structure represented by formula (B), the N at positions 4, 6, 8, 9 and 12 counting from the 5' end of the antisense strand are M These are 2'-O-methyl modified nucleotides, and are located at positions 7 and 16 from the 5' end of the antisense strand. M These are 2'-fluoromodified nucleotides, respectively. In certain other embodiments in which the RNAi construct includes a structure represented by formula (B), the N at positions 7, 8, 9 and 12, counting from the 5' end of the antisense strand, are present. M These are 2'-O-methyl modified nucleotides, and are located at positions 4, 6, and 16 of the antisense strand, counting from the 5' end. M These are 2'-fluoromodified nucleotides, respectively. In these and other embodiments, the RNAi construct includes a structure represented by formula (B), in which N in the sense strand M This is a 2'-fluoromodified nucleotide. In an alternative embodiment, N in the sense strand M This is a 2'-O-methyl modified nucleotide.

[0068] In any of the above embodiments, the RNAi construct includes a structure represented by formula (B), in which each N in both the sense strand and the antisense strand. LThis can be a 2'-O-methyl modified nucleotide. In these embodiments and in any of the embodiments described above, N in formula (B) T This may be an inverted debasic nucleotide, an inverted deoxyribonucleotide, or a 2'-O-methyl modified nucleotide.

[0069] The RNAi constructs of the present invention may also include one or more modified nucleotide bonds. As used herein, the term “modified nucleotide bond” refers to a nucleotide bond other than the natural 3'-5' phosphodiester bond. In some embodiments, the modified nucleotide bond is a phosphorus-containing nucleotide bond such as phosphotriesters, aminoalkyl phosphotriesters, alkylphosphonates (e.g., methylphosphonate, 3'-alkylenephosphonate), phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidates and aminoalkylphosphoramidates), phosphorothioates, chiral phosphorothioates, phosphorodithioates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In one embodiment, the modified nucleotide bond is a 2'-5' phosphodiester bond. In other embodiments, the modified nucleotide bond is a phosphorus-free nucleotide bond and may therefore be called a modified nucleoside bond. Examples of such phosphorus-free bonds include, but are not limited to, morpholino bonds (partially formed from the sugar portion of a nucleoside); siloxane bonds (-O-Si(H)2-O-); sulfide, sulfoxide, and sulfone bonds; formacetyl and thioformacetyl bonds; alkene-containing skeletons; sulfamic acid skeletons; methylene methylimino (-CH2-N(CH3)-O-CH2-) and methylene hydrazino bonds; sulfonic acid and sulfonamide bonds; amide bonds; and others having mixed N, O, S, and CH2 constituent parts. In one embodiment, the modified internucleoside bond is a peptide-based bond (e.g., aminoethylglycine) for generating peptide nucleic acids or PNAs, such as those described in U.S. Patents No. 5,539,082, No. 5,714,331, and No. 5,719,262.Other suitable modified nucleotide-nucleotide and nucleoside-nucleotide bonds that may be employed in the RNAi construct of the present invention are all described in U.S. Patent No. 6,693,187, U.S. Patent No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761 and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, which are all incorporated herein by reference.

[0070] In certain embodiments, the RNAi construct of the present invention includes one or more phosphorothioate nucleotide interbonds. These phosphorothioate nucleotide interbonds may be present on the sense strand, antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand includes one, two, three, four, five, six, seven, eight or more phosphorothioate nucleotide interbonds. In other embodiments, the antisense strand includes one, two, three, four, five, six, seven, eight or more phosphorothioate nucleotide interbonds. In yet another embodiment, both strands include one, two, three, four, five, six, seven, eight or more phosphorothioate nucleotide interbonds. The RNAi construct may include one or more phosphorothioate nucleotide interbonds at the 3' end, 5' end, or both the 3' and 5' ends of the sense strand, antisense strand, or both strands. For example, in certain embodiments, the RNAi construct includes about one to about six or more (e.g., about one, two, three, four, five, six or more) consecutive phosphorothioate nucleotide bonds at the 3' ends of the sense strand, antisense strand, or both strands. In other embodiments, the RNAi construct includes about one to about six or more (e.g., about one, two, three, four, five, six or more) consecutive phosphorothioate nucleotide bonds at the 5' ends of the sense strand, antisense strand, or both strands. In a particular embodiment, the antisense strand includes at least one but six or fewer phosphorothioate nucleotide bonds, and the sense strand includes at least one but four or fewer phosphorothioate nucleotide bonds. In another particular embodiment, the antisense strand includes at least one but four or fewer phosphorothioate nucleotide bonds, and the sense strand includes at least one but two or fewer phosphorothioate nucleotide bonds.

[0071] In some embodiments, the RNAi construct includes a single phosphorothioate nucleotide linkage between the 3' terminal nucleotides of the sense strand. In other embodiments, the RNAi construct includes two consecutive phosphorothioate nucleotide linkages between the 3' terminal nucleotides of the sense strand. In one embodiment, the RNAi construct includes a single phosphorothioate nucleotide linkage between the 3' terminal nucleotides of the sense strand and a single phosphorothioate nucleotide linkage between the 3' terminal nucleotides of the antisense strand. In another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide linkages between the 3' terminal nucleotides of the antisense strand (i.e., phosphorothioate nucleotide linkages between the first and second nucleotide linkages at the 3' end of the antisense strand). In yet another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide linkages between the 3' and 5' terminal nucleotides of the antisense strand. In another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide links between both the 3' and 5' terminal nucleotides of the antisense strand, and two consecutive phosphorothioate nucleotide links at the 5' end of the sense strand. In yet another embodiment, the RNAi construct includes two consecutive phosphorothioate nucleotide links between both the 3' and 5' terminal nucleotides of the antisense strand, and two consecutive phosphorothioate nucleotide links between the terminal nucleotides at the 3' end of the sense strand. In another embodiment, the RNAi construct includes two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends of the antisense strand, and also includes two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends of the sense strand (i.e., phosphorothioate internucleotide bonds between the first and second internucleotide bonds at both the 5' and 3' ends of the antisense strand, and phosphorothioate internucleotide bonds between the first and second internucleotide bonds at both the 5' and 3' ends of the sense strand).In another embodiment, the RNAi construct includes two consecutive phosphorothioate internucleotide bonds between both the 3' and 5' terminal nucleotides of the antisense strand, and a single phosphorothioate internucleotide bond between the terminal nucleotides of the 3' terminal of the sense strand. In any embodiment in which one or both strands include one or more phosphorothioate internucleotide bonds, the remaining internucleotide bonds in the strand may be native 3'-5' phosphodiester bonds. For example, in some embodiments, each internucleotide bond in the sense and antisense strands is selected from phosphodiesters and phosphorothioates, and at least one internucleotide bond is a phosphorothioate.

[0072] In embodiments where the RNAi construct includes a nucleotide overhang, two or more unpaired nucleotides within the overhang may be linked by phosphorothioate nucleotide linkages. In certain embodiments, all unpaired nucleotides within the nucleotide overhang at the 3' end of the antisense and / or sense strand are linked by phosphorothioate nucleotide linkages. In other embodiments, all unpaired nucleotides within the nucleotide overhang at the 5' end of the antisense and / or sense strand are linked by phosphorothioate nucleotide linkages. In yet another embodiment, all unpaired nucleotides within any nucleotide overhang are linked by phosphorothioate nucleotide linkages.

[0073] The incorporation of phosphorothioate nucleotide interbonding introduces an additional chiral center at the phosphorus atom of the oligonucleotide, thus generating a diastereomer pair (Rp and Sp) at each phosphorothioate nucleotide interbonding. Diastereomers or diastereoisomers are different stereoconfigurations of compounds that have the same molecular formula and sequence of the atoms being bonded, but differ in the three-dimensional orientation of those atoms in space. Unlike enantiomers, diastereomers are not mirror images of each other. Each chiral phosphate atom can be in the "R" configuration (Rp) or the "S" configuration (Sp). In certain embodiments, the RNAi construct of the present invention may include one or more phosphorothioate nucleotide interbondings, selected such that the chiral phosphate is primarily in either the Rp or Sp configuration. For example, in some embodiments where the RNAi construct has one or more phosphorothioate nucleotide interbondings, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the chiral phosphate is in the Sp configuration. In other embodiments where the RNAi construct has one or more phosphorothioate nucleotide interlinks, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the chiral phosphates are in the Rp configuration. All chiral phosphates in the RNAi construct can be either in the Sp configuration or the Rp configuration (i.e., the RNAi construct is stereopure). In one particular embodiment, all chiral phosphates in the RNAi construct are in the Sp configuration. In another particular embodiment, all chiral phosphates in the RNAi construct are in the Rp configuration.

[0074] In certain embodiments, the chiral phosphate in the RNAi construct may have different configurations at different positions in the sense strand or antisense strand. In one such embodiment in which the RNAi construct includes one or two phosphorothioate nucleotide interbonds at the 5' end of the antisense strand, the chiral phosphate at the 5' end of the antisense strand may be in an Rp configuration. In another such embodiment in which the RNAi construct includes one or two phosphorothioate nucleotide interbonds at the 3' end of the antisense strand, the chiral phosphate at the 3' end of the antisense strand may be in an Sp configuration. In certain embodiments, the RNAi construct comprises two consecutive phosphorothioate nucleotide links between both the 3' and 5' terminal nucleotides of the antisense strand and two consecutive phosphorothioate nucleotide links between the terminal nucleotides of the 3' terminal of the sense strand, wherein the chiral phosphate at the 5' terminal of the antisense strand is in the Rp configuration, the chiral phosphate at the 3' terminal of the antisense strand is in the Sp configuration, and the chiral phosphate at the 3' terminal of the sense strand may be in either the Rp or Sp configuration. In certain other embodiments, the RNAi construct comprises two consecutive phosphorothioate nucleotide links between both the 3' and 5' terminal nucleotides of the antisense strand and a single phosphorothioate nucleotide link between the terminal nucleotides of the 3' terminal of the sense strand, wherein the chiral phosphate at the 5' terminal of the antisense strand is in the Rp configuration, the chiral phosphate at the 3' terminal of the antisense strand is in the Sp configuration, and the chiral phosphate at the 3' terminal of the sense strand may be in either the Rp or Sp configuration.Methods for controlling the stereochemistry of phosphorothioate bonds during oligonucleotide synthesis are known to those skilled in the art, and include those described in Nawrot and Rebowska, Curr Protoc Nucleic Acid Chem. 2009, Chapter 4:.doi:10.1002 / 0471142700.nc0434s362009; Jahns et al., Nat.Commun, Vol.6:6317, 2015; Knouse et al., Science, Vol.361:1234-1238, 2018; and Sakamuri et al., Chembiochem, Vol.21(9):1304-1308, 2020.

[0075] In some embodiments of the RNAi construct of the present invention, the 5' ends of the sense strand, antisense strand, or both the antisense strand and the sense strand include a phosphate moiety. As used herein, the term “phosphate moiety” refers to terminal phosphate groups including unmodified phosphate (-OP=O)(OH)OH) and modified phosphates. Modified phosphates include O and OH groups, one or more of which are H, O, S, N(R) or alkyl (e.g., C1-C1). 12 ) is substituted with, and R is H, an amino protecting group or an unsubstituted or substituted alkyl (for example, C1~C 12 The phosphate moieties include, but are not limited to, 5'-monophosphates; 5'-diphosphates; 5'-triphosphates; 5'-guanosine caps (7-methylated or unmethylated); 5'-adenosine caps or any other modified or unmodified nucleotide cap structures; 5'-monothiophosphates (phosphorothioates); 5'-monodhithiophosphates (phosphorodithioates); 5'-α-thiotriphosphates; 5'-γ-thiotriphosphates; 5'-phosphoamidates; 5'-vinylphosphates; 5'-alkylphosphonates (e.g., alkyl=methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyletherphosphonates (e.g., alkylether=methoxymethyl, ethoxymethyl, etc.).

[0076] Modified nucleotides that can be incorporated into the RNAi construct of the present invention may have two or more chemical modifications as described herein. For example, a modified nucleotide may have modifications to a ribose sugar and modifications to a nucleic acid base. For example, a modified nucleotide may include a 2' sugar modification (e.g., 2'-fluoro or 2'-O-methyl) and a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, a modified nucleotide may include a sugar modification combined with a modification to a 5' phosphate, which will create a modified nucleotide-to-modified bond or a nucleoside bond when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, a modified nucleotide may include sugar modifications such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification and a 5' phosphorothioate group. Thus, in some embodiments, one or both strands of the RNAi construct of the present invention include a 2' modified nucleotide or a combination of BNA and a phosphorothioate nucleotide-to-nucleotide bond. In certain embodiments, both the sense and antisense strands of the RNAi construct of the present invention include a combination of 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, and phosphorothioate nucleotide interbondings. Exemplary RNAi constructs including modified nucleotides and modified nucleotide interbondings are shown in Table 2.

[0077] The RNAi construct of the present invention can be readily prepared using techniques known in the art, for example, by conventional solid-phase nucleic acid synthesis methods. The polynucleotides of the RNAi construct can be constructed using standard nucleotides or nucleoside precursors (e.g., phosphoramidites) in a suitable nucleic acid synthesizer. Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA). An exemplary method for synthesizing the RNAi construct of the present invention is described in Example 2.

[0078] Oligonucleotides can be synthesized by phosphoramidite chemistry using a 2'-silyl protecting group in combination with dimethoxytrityl (DMT), which is acid-unstable at the 5' position of the ribonucleoside. The final deprotection conditions are known not to significantly degrade the RNA product. All synthesis can be carried out on any automated or manual synthesizer, large, medium, or small scale. Synthesis can also be performed in multi-well plates, columns, or glass slides.

[0079] The 2'-O-silyl group can be removed by exposure to fluoride ions, which may include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride. Crown ether catalysts can be used in combination with inorganic fluorides in the deprotection reaction. Exemplary sources of fluoride ions are tetrabutylammonium fluoride or amine hydrofluorides (e.g., triethylamine combined with aqueous HF in a dipolar aproton solvent, such as dimethylformamide).

[0080] The stability of tryesters with respect to fluorides can be altered by selecting the appropriate protecting group for use with phosphite tryesters and phosphotryesters. Methyl protection of phosphotryesters or phosphite tryesters can stabilize their binding to fluoride ions and improve process yields.

[0081] Since ribonucleosides have a reactive 2'-hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group (for example, a protecting group that is stable to acid treatment). Silyl protecting groups satisfy this condition and can be easily removed in the final fluoride deprotection step, thereby minimizing RNA degradation.

[0082] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Exemplary catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0083] As can be understood by those skilled in the art, further methods for synthesizing the RNAi constructs described herein will be apparent to them. In addition, various synthetic steps can be carried out in alternative order or sequence to obtain the desired compounds. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc., present in bases), and methods for protecting and deprotecting the RNAi constructs described herein that are useful in the synthesis of the RNAi constructs described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW. Greene and PGMWuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof. Custom synthesis of RNAi constructs is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0084] The RNAi constructs of the present invention may include ligands. As used herein, “ligand” means any compound or molecule that can interact directly or indirectly with another compound or molecule. The interaction between another compound or molecule and a ligand may induce a biological response (e.g., triggering a signaling cascade, inducing receptor-mediated endocytosis) or may be a physical association. A ligand can modify one or more properties of the double-stranded RNA molecule it binds to, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, intracellular uptake, charge, and / or clearance properties of the RNA molecule.

[0085] Ligands include serum proteins (e.g., human serum albumin, low-density lipoprotein, globulin), cholesterol moieties, and vitamins (biotin, vitamin E, vitamin B). 12Ligands may include folic acid moieties, steroids, bile acids (e.g., cholic acid), fatty acids (e.g., palmitic acid, myristic acid), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), glycosides, phospholipids, or antibodies or their binding fragments (e.g., antibodies or binding fragments that target RNAi constructs against specific cell types such as liver). Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecyl It contains glycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptides (e.g., Antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers (e.g., polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids and polyamines (e.g., spermine, spermidine).

[0086] In certain embodiments, the ligand has endosomal destabilizing properties. The endosomal destabilizing ligand promotes the lysis of endosomes and / or the transport of the RNAi construct or its components from endosomes to the cytoplasm of cells. The endosomal destabilizing ligand may be a polycationic peptide or peptide mimetic exhibiting pH-dependent membrane activity and membrane fusion properties. In one embodiment, the endosomal destabilizing ligand adopts its active conformation at the pH of endosomes. The "active" conformation is the conformation in which the endosomal destabilizing ligand promotes the lysis of endosomes and / or the transport of the RNAi construct or its components from endosomes to the cytoplasm of cells. Examples of endosome destabilizing ligands include GALA peptide (Subbarao et al., Biochemistry, Vol.26:2964-2972, 1987), EALA peptide (Vogel et al., J.Am.Chem.Soc., Vol.118:1581-1586, 1996), and their derivatives (Turk et al., Biochem.Biophys.Acta, Vol.1559:56-68, 2002). In one embodiment, the endosome destabilizing component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosome destabilizing component may be linear or branched.

[0087] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or another steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Patent Nos. 7,851,615; U.S. Patent Nos. 7,745,608; and U.S. Patent Nos. 7,833,992, all of which are incorporated herein by reference in their entirety. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to a folate moiety can be taken up by cells via receptor-dependent endocytosis pathways. Such folate-polynucleotide conjugates are described in U.S. Patent No. 8,188,247, which is incorporated herein by reference in its entirety.

[0088] In certain embodiments, it is desirable to specifically deliver the RNAi construct of the present invention to hepatocytes to specifically reduce the expression of the mARC1 protein in the liver. Therefore, in certain embodiments, ligands are targeted for specific delivery of the RNAi construct to hepatocytes (e.g., hepatocytes) by various means, as will be described in more detail below. In certain embodiments, the RNAi construct is targeted to hepatocytes by ligands that bind to surface-expressed asialoglycoprotein receptors (ASGRs) or their components (e.g., ASGR1, ASGR2).

[0089] In some embodiments, the RNAi construct can be specifically targeted to the liver by employing ligands that bind to or interact with proteins expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand may include an antigen-binding protein (e.g., an antibody or its binding fragment (e.g., Fab, scFv)) that specifically binds to receptors expressed on hepatocytes, such as the asialoglycoprotein receptor and the LDL receptor. In one particular embodiment, the ligand includes an antibody or its binding fragment that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. The "Fab fragment" consists of one immunoglobulin light chain (i.e., the light chain variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. In another embodiment, the ligand includes a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. The “scFv fragment” comprises a VH region and a VL region of an antibody, which are present in a single polypeptide chain and optionally include a peptide linker between the VH and VL regions that allows Fv to form a desired structure for antigen binding. Exemplary antibodies and their conjugated fragments that specifically bind to ASGR1 and can be used as ligands for targeting the RNAi construct of the present invention to the liver are described in their entirety in International Publication No. 2017 / 058944, which is incorporated herein by reference. Other antibodies or their conjugated fragments that specifically bind to ASGR1, the LDL receptor, or other proteins expressed on the surface of the liver and are suitable for use as ligands in the RNAi construct of the present invention are commercially available.

[0090] In certain embodiments, the ligand includes carbohydrates. “Carbohydrate” refers to a compound composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) with oxygen, nitrogen, or sulfur atoms bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrates incorporated into the ligand are monosaccharides selected from pentoses, hexoses, or heptoses, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrates incorporated into the ligand are amino sugars, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0091] In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting the compound to hepatocytes because such ligands bind to ASGR expressed on the surface of hepatocytes. For example, see D'Souza and Devarajan, J. Control Release, Vol. 203: 126-139, 2015. Examples of GalNAc or galactose-containing ligands that can be incorporated into the RNAi construct of the present invention are described in U.S. Patent No. 7,491,805; No. 8,106,022; and No. 8,877,917; U.S. Patent Publication No. 20030130186; and International Publication No. 2013166155, all of which are incorporated herein by reference in their entirety.

[0092] In certain embodiments, the ligand includes a polyhydric carbohydrate moiety. As used herein, “polyhydric carbohydrate moiety” refers to a moiety comprising two or more carbohydrate units that can independently bind to or interact with other molecules. For example, a polyhydric carbohydrate moiety includes two or more binding domains composed of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The valency of a carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety. For example, the terms “monovalent,” “divalent,” “trivalent,” and “tetravalent” with respect to carbohydrate moieties refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A polyhydric carbohydrate moiety may include a polyhydric lactose moiety, a polyhydric galactose moiety, a polyhydric glucose moiety, a polyhydric N-acetyl-galactosamine moiety, a polyhydric N-acetyl-glucosamine moiety, a polyhydric mannose moiety, or a polyhydric fucose moiety. In some embodiments, the ligand includes a polyhydric galactose moiety. In other embodiments, the ligand includes a polyhydric N-acetyl-galactosamine moiety. In these and other embodiments, the polyhydric carbohydrate moiety may be divalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety may be bivalent or trivalent. In one particular embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent or tetravalent GalNAc-containing ligands for incorporation into the RNAi construct of the present invention are detailed below.

[0093] Ligands can be directly or indirectly bound to or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is covalently bound directly to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently bound to the sense or antisense strand of the RNAi construct via a linker. Ligands can be bound to nucleic acid bases, sugar moieties, or internucleotide bonds of the polynucleotide (e.g., sense or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to purine nucleic acid bases or their derivatives can occur at any position, including atoms inside and outside the ring. In certain embodiments, the ligand is bound to positions 2, 6, 7, or 8 of the purine nucleic acid base. Conjugation or binding to pyrimidine nucleic acid bases or their derivatives can also occur at any position. In some embodiments, the ligand can be bound to positions 2, 5, and 6 of the pyrimidine nucleic acid base. Conjugation or binding to the sugar moiety of a nucleotide can occur at any carbon atom. Exemplary carbon atoms in the sugar moiety that can bind to a ligand include the 2', 3', and 5' carbon atoms. For example, in debasalized nucleotides, the 1' position can also bind to a ligand. Nucleotide-nucleotide bonds can also support ligand binding. In the case of phosphorus-containing bonds (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates, etc.), the ligand can bind directly to the phosphorus atom or to the O, N, or S atom bonded to the phosphorus atom. In amine-containing nucleoside bonds or amide-containing nucleoside bonds (e.g., PNA), the ligand can bind to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0094] In some embodiments, the ligand may be bound to the 3' or 5' end of either the sense strand or the antisense strand. In certain embodiments, the ligand is covalently bound to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5' terminal nucleotide of the sense strand. In these embodiments and other embodiments, the ligand is attached at the 5' position of the 5'-terminal nucleotide of the sense strand. In embodiments where the inverted debasalized nucleotide is the 5' terminal nucleotide of the sense strand and is bound to an adjacent nucleotide via a 5'-5' nucleotide bond, the ligand may be bound to the 3' position of the inverted debasalized nucleotide. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3' position of the 3' terminal nucleotide of the sense strand. In embodiments where the inverted debasalized nucleotide is the 3' terminal nucleotide of the sense strand and is bound to an adjacent nucleotide via a 3'-3' nucleotide bond, the ligand may be bound to the 5' position of the inverted debasalized nucleotide. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but prior to one or more terminal nucleotides (i.e., prior to terminal 1, 2, 3, or 4). In some embodiments, the ligand is attached at the 2' position of the sugar of the 3' terminal nucleotide of the sense strand. In other embodiments, the ligand is attached at the 2' position of the sugar of the 5' terminal nucleotide of the sense strand.

[0095] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches the ligand to the polynucleotide component of the RNAi construct. The linker can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker can generally include a bifunctional linking moiety that includes an alkyl portion having two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g., the sense or antisense strand of the RNAi construct), and the other is selected to bind substantially to any selected group such as a ligand as described herein. In certain embodiments, the linker includes a chain structure or oligomer consisting of repeating units such as ethylene glycol units or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, and unsaturation (e.g., double bond or triple bond), among others.

[0096] Linkers that can be used to attach the ligand to the sense or antisense strand in the RNAi constructs of the invention include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C 10 alkyl, substituted or unsubstituted C2-C 10 alkenyl or substituted or unsubstituted C2-C 10It includes, but is not limited to, alkynyl. Suitable substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0097] In certain embodiments, the linker is cleavable. A cleavable linker is sufficiently stable outside the cell but is cleaved after entry into the target cell to release the two portions that the linker holds together. In some embodiments, the cleavable linker is cleaved at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold faster or at least 100-fold faster in the target cell or under a first reference condition (e.g., selected to mimic or correspond to intracellular conditions) than in the subject's blood or under a second reference condition (e.g., selected to mimic or correspond to conditions found in blood or serum).

[0098] A cleavable linker is sensitive to a cleaving agent, such as the presence of pH, redox potential, or a degradable molecule. Generally, the cleaving agent is predominant or found at a high level or activity inside the cell rather than in serum or blood. Examples of such degradable agents include, for example, oxidases or reductases or reducing agents, such as mercaptans, that are present inside the cell and can cleave a redox-cleavable linker by reduction; esterases; agents that can generate an endosome or an acidic environment, such as those that bring about a pH of 5 or less; enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or degrade an acid-cleavable linker by acting as a general acid.

[0099] Cleavable linkers may contain pH-sensitive regions. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable groups that are cleaved at a favorable pH, thereby releasing RNA molecules from the ligand into the cell interior or a desired compartment of the cell.

[0100] A linker may contain cleavable groups that can be cleaved by specific enzymes. The type of cleavable groups incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand may bind to an RNA molecule via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore the linker will be cleaved more efficiently in hepatocytes than in esterase-deficient cell types. Other types of cells rich in esterases include lung, renal cortex, and testicular cells. When targeting peptidase-rich cells such as hepatocytes and synovial cells, linkers containing peptide bonds can be used.

[0101] Generally, the suitability of a cleavable linker candidate can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the linker candidate. It is also desirable to test the cleavable linker candidate for its ability to resist cleavage in blood or in contact with other non-target tissues. Therefore, relative susceptibility to cleavage can be determined between a first condition selected to exhibit cleavage within target cells and a second condition selected to exhibit cleavage in other tissues or body fluids, such as blood or serum. Evaluation can be performed in cell-free systems, cells, cell cultures, organs or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and then confirm it by further evaluation in whole animals. In some embodiments, a useful linker candidate cleaves at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0102] In other embodiments, redox-cleavable linkers are utilized. Redox-cleavable linkers are cleaved when reduced or oxidized. An example of a reductively cleavable group is a disulfide linking group (-SS-). One or more of the methods described herein can be used to determine whether a cleavable linker candidate is a suitable “reductively cleavable linker” or suitable for use with, for example, a particular RNAi construct and a particular ligand. For example, a linker candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate that would be observed in cells such as target cells. Linker candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the linker candidate is cleaved up to 10% in blood. In other embodiments, useful linker candidates are degraded at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0103] In yet another embodiment, a ligand is covalently bonded to the sense or antisense strand of an RNAi construct using a phosphate-based cleavable linker that is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that hydrolyzes the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-cleavable groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S- and -OP(S)(Rk)-S-, where Rk is hydrogen or C1~C 10It can be alkyl. Specific embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. Another specific embodiment is -OP(O)(OH)-O-. These linker candidates can be evaluated using methods similar to those described above.

[0104] In other embodiments, the linker may include an acid-cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or below) or by a drug such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for the acid-cleavable group. Examples of acid-cleavable binding groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable group may have the general formula -C=NN-, C(O)O, or -OC(O). In certain embodiments, the carbon bonded to the oxygen (alkoxy group) of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethyl, pentyl, or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0105] In other embodiments, the linker may contain ester-based cleavable groups that are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable groups have the general formula -C(O)O- or -OC(O)-. These linker candidates can be evaluated using methods similar to those described above.

[0106] In further embodiments, the linker may include a cleavable group of a peptide system that is cleaved by enzymes such as peptidases and proteases in cells. The cleavable group of a peptide system is a peptide bond formed between amino acids, such as those that produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group of a peptide system includes an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The cleavable group of a peptide system is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins. The cleavable linking group of a peptide system has the general formula -NHCHR A C(O)NHCHR B It contains C(O)-, in the formula, R A and R B These are the side chains of two adjacent amino acids. These candidates can be evaluated using a method similar to that described above.

[0107] Other types of linkers suitable for binding ligands to the sense or antisense strand in the RNAi construct of the present invention are known in the art and may include the linkers described in U.S. Patent Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entirety.

[0108] In certain embodiments, the ligand covalently bound to the sense or antisense strand of the RNAi construct of the present invention comprises a GalNAc moiety, for example, a polyvalent GalNAc moiety. In some embodiments, the polyvalent GalNAc moiety is a trivalent GalNAc moiety bound to the 3' end of the sense strand. In other embodiments, the polyvalent GalNAc moiety is a trivalent GalNAc moiety bound to the 5' end of the sense strand. In yet another embodiment, the polyvalent GalNAc moiety is a tetravalent GalNAc moiety bound to the 3' end of the sense strand. In yet another embodiment, the polyvalent GalNAc moiety is a tetravalent GalNAc moiety bound to the 5' end of the sense strand.

[0109] In certain embodiments, the RNAi construct of the present invention comprises a ligand having the following structure ([Structure 1]). [ka]

[0110] In a preferred embodiment, a ligand having this structure is covalently bonded to the 5' end of the sense strand (for example, to the 5' terminal nucleotide of the sense strand) via a linker such as a linker described herein. In one embodiment, the linker is an aminohexyl linker.

[0111] Examples of trivalent and tetravalent GalNAc moieties and linkers that can bind to double-stranded RNA molecules in the RNAi construct of the present invention are provided in the following structural formulas I to IX. In the formulas listed herein, "Ac" represents an acetyl group.

[0112] In one embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula I, where each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, and j is 1 or 2, and the ligand is bound to the 3' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0113] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula II below, wherein each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line).

Chemical formula

[0114] In still another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula III below, wherein the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line).

Chemical formula

[0115] In yet another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula IV below, wherein the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line).

Chemical formula

[0116] In a particular embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula V below, wherein each n is independently 1 to 3, k is 1 to 3, and the ligand is attached to the 5' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line).

Chemical formula

[0117] In other embodiments, the RNAi construct comprises a ligand and a linker having the structure of formula VI below, where each n is independently 1 to 3 and k is 1 to 3, and the ligand is bound to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0118] In one particular embodiment, the RNAi construct comprises a ligand and a linker having the structure of the following formula VII, where X = O or S, and the ligand is bound to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a wavy line). [ka]

[0119] In some embodiments, the RNAi construct comprises a ligand and a linker having the structure of formula VIII below, where n is independently 1 to 3, and the ligand is bound to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0120] In certain embodiments, the RNAi construct comprises a ligand and a linker having the structure of formula IX, wherein the ligand is bound to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid dashed line). [ka]

[0121] The phosphorothioate bond can be replaced with a phosphodiester bond, as shown in any one of formulas I to IX, in order to covalently bond the ligand and linker to the nucleic acid chain.

[0122] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi construct described herein and pharmaceutically acceptable carriers, excipients, or diluents. Such compositions and formulations are useful for reducing the expression of the MARC1 gene in patients in need. Where clinical use is envisioned, the pharmaceutical compositions and formulations are prepared in a form appropriate for the intended use. Generally, this involves preparing compositions that are substantially free not only of pyrogens but also of other impurities that may be harmful to humans or animals.

[0123] The terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse allergic or other undesirable reactions when administered to animals or humans. As used herein, “pharmaceutically acceptable carriers, excipients or diluents” include solvents, buffers, solutions, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are acceptable for use in the formulation of pharmaceuticals, such as drugs suitable for administration to humans. The use of such media and drugs for pharmaceutically active substances is known in the art. Any conventional media or drugs are intended for use in therapeutic compositions unless they are incompatible with the RNAi construct of the present invention. Auxiliary active ingredients may also be incorporated into the composition, provided they do not inactivate the RNAi construct of the composition.

[0124] The composition and method of formulation of a pharmaceutical composition depend on several conditions, including but not limited to the route of administration, the type and degree of the disease or disorder being treated, or the dose administered. In some embodiments, the pharmaceutical composition is formulated based on the intended route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include intravenous, intra-arterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may comprise a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may comprise a targeted ligand (e.g., a GalNAc-containing or antibody-containing ligand as described herein).

[0125] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi construct described herein. “Effective amount” is an amount sufficient to produce a favorable or desired clinical outcome. In some embodiments, the effective amount is sufficient to reduce the expression of the MARC1 gene in a particular tissue or cell type of the patient (e.g., liver or hepatocytes). An effective amount of the RNAi construct of the present invention may range from about 0.01 mg / kg body weight to about 100 mg / kg body weight and may be administered daily, weekly, monthly, or at longer intervals. Precisely determining what is considered an effective dose and frequency may be based on several factors, including the patient's height, age, and overall health, the type of disorder being treated (e.g., fatty liver disease, hepatic fibrosis, or cardiovascular disease), the specific RNAi construct used, and the route of administration.

[0126] The pharmaceutical composition of the present invention may be administered via any common route, provided that the target tissue is available through that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or direct injection into liver tissue or delivery via the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0127] Colloidal dispersion systems, such as oil-in-water emulsions, micelles, mixed micelles and liposomes, polymer complexes, nanocapsules, microspheres, beads and lipid-based systems, can be used as delivery vehicles for the RNAi constructs of the present invention. Suitable commercially available lipid emulsions for delivering the nucleic acids of the present invention include Intralipid® (Baxter International Inc.), Liposyn® (Abbott Pharmaceuticals), Liposyn® II (Hospira), Liposyn® III (Hospira), Nutrilipid (B. Braun Medical Inc.) and other similar lipid emulsions. An exemplary colloidal system for in vivo use as a delivery vehicle is liposomes (i.e., artificial membrane vesicles). The RNAi constructs of the present invention can be encapsulated within liposomes or can form complexes with liposomes, particularly cationic liposomes. In addition, the RNAi constructs of the present invention can form complexes with lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersions are well known in the art. Exemplary formulations are disclosed in U.S. Patent Nos. 5,981,505, 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and in International Publication No. 03 / 093449.

[0128] In some embodiments, the RNAi constructs of the present invention are completely encapsulated, for example, in lipid formulations to form SNALPs or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles. SNALPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs are extremely useful for systemic administration because they exhibit a long circulating lifetime after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site). Nucleic acid-lipid particles typically have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm and are substantially non-toxic. In addition, nucleic acids present in nucleic acid-lipid particles are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, and 6,815,432, and in International Publication No. 96 / 40964.

[0129] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile solutions or dispersions for injection. Generally, these preparations are sterile and fluid enough to be easily injected. The preparations must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. For example, by using a coating such as lecithin, the particle size required in the case of dispersions can be maintained, and by using a surfactant, appropriate fluidity can be maintained. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injection composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0130] Sterile injectable solutions can be prepared by incorporating an appropriate amount of the active compound into a solvent, along with any other optional components as needed (e.g., those listed above), and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other desired components, such as those listed above. For sterile powders used to prepare sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, from which powders of the active ingredient plus any additional desired components are obtained from their pre-sterilized filtered solutions.

[0131] The compositions of the present invention can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with a free amino group) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, and mandelic acid). Salts formed with a free carboxyl group can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, and procaine). Pharmaceutically acceptable salts are described in detail in Berge et al., J. Pharmaceutical Sciences, Vol. 66: 1-19, 1977.

[0132] For parenteral administration in aqueous solutions, for example, the solution is usually appropriately buffered, and the liquid diluent is first isotonic with, for example, sufficient saline or glucose. Such aqueous solutions can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Particularly in consideration of this disclosure, it is preferable to use sterile aqueous media as known to those skilled in the art. As an example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the indicated injection site (see, for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). When administered to humans, the preparation must meet the sterility, pyrogenicity, general safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises or consists of sterile saline and the RNAi construct described herein. In other embodiments, the pharmaceutical composition of the present invention comprises or consists of the RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet another embodiment, the pharmaceutical composition of the present invention comprises or consists of the RNAi construct described herein and phosphate-buffered saline (PBS).

[0133] In some embodiments, the pharmaceutical compositions of the present invention are packaged in or stored within an administration device. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, autoinjectors, injection pumps, attachable syringes, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to, inhalers, inhalers, and aspirators. Accordingly, the present invention includes an administration device containing a pharmaceutical composition of the present invention for treating or preventing one or more of the disorders or diseases described herein.

[0134] The present invention provides a method for reducing or inhibiting the expression of the MARC1 gene, and therefore the production of the mARC1 protein, in cells (e.g., hepatocytes) by contacting cells with any one of the RNAi constructs described herein. The cells may be in vitro or in vivo. mARC1 expression can be assessed by measuring the amount or level of another biomarker associated with mARC1 expression, such as the mRNA of mARC1, the protein of mARC1, or serum levels of cholesterol, LDL cholesterol, or liver enzymes, such as alanine aminotransferase (ALT). The reduction in mARC1 expression in cells or animals treated with the RNAi construct of the present invention can be determined by comparing it to mARC1 expression in cells or animals that have not been treated with the RNAi construct or have been treated with a control RNAi construct. For example, in some embodiments, the reduction in mARC1 expression is evaluated by (a) measuring the amount or level of mARC1 mRNA in hepatocytes treated with the RNAi construct of the present invention, (b) measuring the amount or level of mARC1 mRNA in hepatocytes treated with a control RNAi construct (e.g., an RNAi construct targeting RNA molecules not expressed in hepatocytes or an RNAi construct having a nonsense or scrambled sequence) or without the construct, and (c) comparing the mARC1 mRNA level measured from the cells treated in (a) with the mARC1 mRNA level measured from the control cells in (b). Before comparison, the mARC1 mRNA levels in the treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). The mRNA level of mARC1 can be measured by a variety of methods, including Northern blotting, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and droplet digital PCR.

[0135] In other embodiments, the reduction of mARC1 expression is evaluated by (a) measuring the amount or level of mARC1 protein in hepatocytes treated with the RNAi construct of the present invention; (b) measuring the amount or level of mARC1 protein in hepatocytes treated with a control RNAi construct (e.g., an RNAi construct targeting RNA molecules not expressed in hepatocytes or an RNAi construct having a nonsense or scrambled sequence) or without the construct; and (c) comparing the mARC1 protein level measured from cells treated in (a) with the mARC1 protein level measured from control cells in (b). Methods for measuring mARC1 protein levels are known to those skilled in the art and include, for example, Western blotting, immunoassays (e.g., ELISA), and flow cytometry. The effectiveness of the RNAi construct of the present invention can be evaluated using any method capable of measuring mARC1 mRNA or mARC1 protein.

[0136] In some embodiments, the method for evaluating the expression level of mARC1 is performed in vitro in cells that naturally express mARC1 (e.g., hepatocytes) or cells that have been engineered to express mARC1. In certain embodiments, the method is performed in vitro in hepatocytes. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human and non-human primate hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the hepatocytes are HuH-7 cells. In another embodiment, the hepatocytes are human primary hepatocytes. In yet another embodiment, the hepatocytes are Hep3B cells.

[0137] In other embodiments, methods for evaluating mARC1 expression levels are performed in vivo. An RNAi construct and any control RNAi construct may be administered to animals, and levels of mARC1 mRNA or mARC1 protein can be evaluated in liver tissue collected from the treated animals. Alternatively, or in addition, biomarkers or functional phenotypes associated with mARC1 expression can be evaluated in the treated animals. For example, loss-of-function variants of MARC1 are associated with reduced serum total cholesterol, LDL cholesterol, and liver enzyme levels (see Emdin et al., PLoS Genet, Vol.16(4):e1008629, 2020). Therefore, serum or plasma levels of cholesterol, LDL cholesterol, or liver enzymes (e.g., ALT) can be measured in animals treated with the RNAi construct of the present invention to evaluate the functional effectiveness of reducing mARC1 expression. Exemplary methods for measuring serum or plasma cholesterol or enzyme levels are described in Examples 1, 4, and 5.

[0138] In certain embodiments, the expression of mARC1 mRNA or protein is reduced by at least 40%, at least 45%, or at least 50% in hepatocytes by the RNAi construct of the present invention. In some embodiments, the expression of mARC1 mRNA or protein is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in hepatocytes by the RNAi construct of the present invention. In other embodiments, the expression of mARC1 mRNA or protein is reduced by about 90% or more in hepatocytes by the RNAi construct of the present invention, for example, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more. The rate of reduction of mARC1 expression can be measured by any of the methods described herein and other methods known in the art.

[0139] The present invention provides a method for reducing or inhibiting the expression of the MARC1 gene, and therefore the production of the mARC1 protein, in patients who require it, and a method for treating or preventing conditions, diseases, or disorders associated with mARC1 expression or activity. “Conditions, diseases, or disorders associated with mARC1 expression” refers to conditions, diseases, or disorders in which the expression level of mARC1 is altered or elevated levels of mARC1 are associated with an increased risk of developing that condition, disease, or disorder. Conditions, diseases, or disorders associated with mARC1 expression may also include conditions, diseases, or disorders resulting from abnormal changes in lipoprotein metabolism, such as changes that lead to abnormal or high levels of cholesterol, lipids, triglycerides, etc., or changes that lead to impaired clearance of these molecules. Recent genetic studies have reported associations between loss-of-function variants in the MARC1 gene and decreased blood levels of cholesterol and liver enzymes, reduced liver fat, and prevention of cirrhosis (Spracklen et al., Hum Mol Genet., Vol.26(9):1770-178, 2017; Emdin et al., bioRxiv 594523; / / doi.org / 10.1101 / 594523, 2019; and Emdin et al., PLoS Genet, Vol.16(4):e1008629, 2020). See Emdin et al., bioRxiv 594523; / / doi.org / 10.1101 / 594523,2019; and Emdin et al., PLoS Genet, Vol.16(4):e1008629,2020. For this reason, in certain embodiments, the RNAi construct of the present invention is particularly useful for the treatment or prevention of fatty liver disease (e.g., NAFLD and NASH) and cardiovascular disease (e.g., coronary artery disease and myocardial infarction), as well as for hepatic fibrosis and the reduction of serum cholesterol levels.

[0140] Conditions, diseases, and disorders associated with mARC1 expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, fatty liver diseases, e.g., alcoholic fatty liver disease, alcoholic steatohepatitis, NAFLD and NASH; chronic liver disease; cirrhosis; cardiovascular diseases, e.g., myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral artery disease), cerebrovascular disease, fragility plaque and aortic stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia (e.g., manifesting as elevated total cholesterol, elevated low-density lipoprotein (LDL), elevated very low-density lipoprotein (VLDL), elevated triglycerides and / or low levels of high-density lipoprotein (HDL)).

[0141] In certain embodiments, the present invention provides a method for reducing the expression of the mARC1 protein in a patient in need thereof, comprising administering one of the RNAi constructs described herein to the patient. As used herein, the term “patient” refers to a mammal, including humans, and can be used interchangeably with the term “subject.” Preferably, the expression level of mARC1 in the patient’s hepatocytes is reduced after administration of the RNAi construct compared to the mARC1 expression level of a patient who has not received the RNAi construct or the mARC1 expression level of a patient before administration of the RNAi construct. In some embodiments, after administration of the RNAi construct of the present invention, mARC1 expression in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The rate of reduction in mARC1 expression can be measured by any of the methods described herein and other methods known in the art. In certain embodiments, the rate of reduction in mARC1 expression is determined by evaluating the levels of serum or plasma biomarkers, such as total cholesterol, LDL cholesterol, or liver enzyme (e.g., ALT) levels in the patient, according to the methods described herein.

[0142] In some embodiments, patients requiring reduction of mARC1 expression are patients at risk of myocardial infarction. Patients at risk of myocardial infarction may have a history of myocardial infarction (e.g., have previously suffered a myocardial infarction). Patients at risk of myocardial infarction may also have a familial history of myocardial infarction or have one or more risk factors for myocardial infarction. Such risk factors include, but are not limited to, hypertension, elevated levels of non-HDL cholesterol, elevated levels of triglycerides, diabetes mellitus, obesity, or a history of autoimmune diseases (e.g., rheumatoid arthritis, lupus). In one embodiment, patients at risk of myocardial infarction are patients with coronary artery disease or diagnosed with coronary artery disease. The risk of myocardial infarction in these and other patients can be reduced by administering any of the RNAi constructs described herein to the patient. Accordingly, the present invention provides a method for reducing the risk of myocardial infarction in patients in need thereof, comprising administering an RNAi construct described herein to the patient. In some embodiments, the present invention includes the use of any of the RNAi constructs described herein in the preparation of a pharmaceutical for reducing the risk of myocardial infarction in patients who require it. In other embodiments, the present invention provides a mARC1-targeted RNAi construct for use in a method for reducing the risk of myocardial infarction in patients who require it.

[0143] In certain embodiments, patients requiring reduction of mARC1 expression are those diagnosed with or at risk of cardiovascular disease. Therefore, the present invention includes methods for treating or preventing cardiovascular disease in patients in need by administering any of the RNAi constructs of the present invention. In some embodiments, the present invention includes the use of any of the RNAi constructs described herein in the preparation of a medicament for treating or preventing cardiovascular disease in patients in need. In other embodiments, the present invention provides a mARC1-targeted RNAi construct for use in methods for treating or preventing cardiovascular disease in patients in need. Cardiovascular diseases include, but are not limited to, myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral artery disease), cerebrovascular disease, fragility plaque, and aortic stenosis. In some embodiments, the cardiovascular disease treated or prevented according to the methods of the present invention is coronary artery disease. In other embodiments, the cardiovascular disease treated or prevented according to the methods of the present invention is myocardial infarction. In yet another embodiment, the cardiovascular disease treated or prevented according to the method of the present invention is stroke. In yet another embodiment, the cardiovascular disease treated or prevented according to the method of the present invention is peripheral artery disease. In certain embodiments, administration of the RNAi construct described herein reduces the risk of non-fatal myocardial infarction, fatal and non-fatal stroke, certain types of cardiac surgery (e.g., angioplasty, bypass), hospitalization for heart failure, chest pain in patients with heart disease and / or cardiovascular events in patients with confirmed heart disease (e.g., history of myocardial infarction, history of cardiac surgery and / or chest pain with signs of arterial occlusion). In some embodiments, administration of the RNAi construct described herein according to the method of the present invention can be used to reduce the risk of recurrent cardiovascular events.

[0144] In some embodiments, the patients treated according to the methods of the present invention are those with fragile plaques (also known as unstable plaques). Fragile plaques are accumulations of macrophages and lipids, primarily cholesterol, beneath the endothelial layer of the arterial wall. These fragile plaques may rupture, potentially leading to the formation of blood clots that can block blood flow through the artery, causing myocardial infarction or stroke. Fragile plaques can be identified by methods known in the art, including but not limited to intravascular ultrasound and computed tomography (see Sahara et al., European Heart Journal, Vol. 25; 2026-2033, 2004; Budhoff, J. Am. Coll. Cardiol., Vol. 48; 319-321, 2006; Hausleiter et al., J. Am. Coll. Cardiol., Vol. 48; 312-318, 2006).

[0145] In other embodiments, patients requiring reduction of mARC1 expression are those with high blood levels of cholesterol (e.g., total cholesterol, non-HDL cholesterol, or LDL cholesterol). Therefore, in some embodiments, the present invention provides a method for reducing blood cholesterol levels (e.g., serum or plasma) in patients requiring such reduction, comprising administering one of the RNAi constructs described herein to the patient. In some embodiments, the present invention includes the use of one of the RNAi constructs described herein in the preparation of a pharmacopoeia for reducing blood cholesterol levels (e.g., serum or plasma) in patients requiring such reduction. In other embodiments, the present invention provides a mARC1-targeted RNAi construct for use in a method for reducing blood cholesterol levels (e.g., serum or plasma) in patients requiring such reduction. In certain embodiments, the cholesterol reduced according to the method of the present invention is LDL cholesterol. In other embodiments, the cholesterol reduced according to the method of the present invention is non-HDL cholesterol. Non-HDL cholesterol is a measure of all cholesterol-containing atherogenic lipoproteins, including LDL cholesterol, very low-density lipoproteins, intermediate-density lipoproteins, lipoprotein(a), chylomicrons, and chylomicron remnants. Non-HDL cholesterol has been reported to be an excellent predictor of cardiovascular risk (Rana et al., Curr. Atheroscler. Rep., Vol. 14: 130-134, 2012). Non-HDL cholesterol levels can be calculated by subtracting HDL cholesterol levels from total cholesterol levels.

[0146] In some embodiments, the patient treated according to the method of the present invention is a patient having elevated levels of non-HDL cholesterol (e.g., elevated serum or plasma levels of non-HDL cholesterol). Ideally, the level of non-HDL cholesterol should be about 30 mg / dL above the target value of LDL cholesterol for any given patient. In certain embodiments, a patient is administered the RNAi construct of the present invention if the patient has a non-HDL cholesterol level of about 130 mg / dL or higher. In one embodiment, a patient is administered the RNAi construct of the present invention if the patient has a non-HDL cholesterol level of about 160 mg / dL or higher. In another embodiment, a patient is administered the RNAi construct of the present invention if the patient has a non-HDL cholesterol level of about 190 mg / dL or higher. In yet another embodiment, a patient is administered the RNAi construct of the present invention if the patient has a non-HDL cholesterol level of about 220 mg / dL or higher. In certain embodiments, a patient is administered the RNAi construct of the present invention if the patient has a high or very high risk of cardiovascular disease according to the 2013 ACC / AHA guideline on the assessment of cardiovascular risk (Goff et al., ACC / AHA guideline on the assessment of cardiovascular risk: a report of the American College of Cardiology / American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol, Vol.63:2935-2959, 2014) and has a non-HDL cholesterol level of approximately 100 mg / dL or higher.

[0147] In certain embodiments of the method of the present invention, a patient is administered the RNAi construct described herein if the patient has a moderate or greater risk of cardiovascular disease according to the 2013 ACC / AHA guidelines for assessing cardiovascular risk (hereinafter referred to as the "2013 Guidelines"). In certain embodiments, the RNAi construct of the present invention is administered to a patient if the patient's LDL cholesterol level is higher than about 160 mg / dL. In other embodiments, the RNAi construct of the present invention is administered to a patient if the patient's LDL cholesterol level is higher than about 130 mg / dL and the patient has a moderate risk of cardiovascular disease according to the 2013 Guidelines. In yet another embodiment, the RNAi construct of the present invention is administered to a patient if the patient's LDL cholesterol level is higher than 100 mg / dL and the patient has a high or very high risk of cardiovascular disease according to the 2013 Guidelines.

[0148] In other embodiments, patients requiring reduction of mARC1 expression are those diagnosed with or at risk of developing fatty liver disease. Therefore, the present invention includes a method for treating, preventing, or reducing the risk of developing fatty liver disease in patients who require such treatment, comprising administering one of the RNAi constructs of the present invention to a patient. In some embodiments, the present invention includes the use of one of the RNAi constructs described herein in the preparation of a pharmacopoeia for treating, preventing, or reducing the risk of developing fatty liver disease in patients who require such treatment. In other embodiments, the present invention provides a mARC1-targeted RNAi construct for use in a method for treating, preventing, or reducing the risk of developing fatty liver disease in patients who require such treatment. Fatty liver disease is a condition characterized by the accumulation of fat in the liver. There are two main types of fatty liver disease: a first type associated with heavy alcohol use (alcoholic steatohepatitis) and a second type not associated with alcohol use (non-alcoholic fatty liver disease (NAFLD)). NAFLD is typically characterized by the presence of fat accumulation in the liver, but with little or no inflammation or hepatocyte damage. NAFLD may progress to non-alcoholic steatohepatitis (NASH), which is characterized by inflammation and cellular damage to the liver, both of which may subsequently lead to hepatic fibrosis and ultimately to cirrhosis or liver cancer. In certain embodiments, the fatty liver disease targeted for treatment, prevention, or reduction of the risk of developing it according to the methods of the present invention is NAFLD. In other embodiments, the fatty liver disease targeted for treatment, prevention, or reduction of the risk of developing it according to the methods of the present invention is NASH. In yet another embodiment, the fatty liver disease targeted for treatment, prevention, or reduction of the risk of developing it according to the methods of the present invention is alcoholic steatohepatitis. In some embodiments, patients who need to be treated or prevented from developing a fatty liver disease according to the methods of the present invention, or who are at risk of developing a fatty liver disease, are diagnosed with type 2 diabetes, or a metabolic disorder, or are obese (e.g., a body mass index of ≥30.0).In other embodiments, patients who require treatment or prevention of fatty liver disease according to the method of the present invention, or patients at risk of developing fatty liver disease, have high levels of non-HDL cholesterol or triglycerides. Depending on the specific patient and other risk factors the patient may have, high levels of non-HDL cholesterol may be about 130 mg / dL or higher, about 160 mg / dL or higher, about 190 mg / dL or higher, or about 220 mg / dL or higher. High triglyceride levels may be about 150 mg / dL or higher, about 175 mg / dL or higher, about 200 mg / dL or higher, or about 250 mg / dL or higher.

[0149] In certain embodiments, patients who require a reduction in mARC1 expression are those diagnosed with or at risk of developing hepatic fibrosis or cirrhosis. Therefore, the present invention encompasses methods for treating, preventing, or reducing hepatic fibrosis in patients who require such treatment, comprising administering one of the RNAi constructs of the present invention to the patient. In some embodiments, the present invention includes the use of one of the RNAi constructs described herein in the preparation of a pharmacopoeia for treating, preventing, or reducing hepatic fibrosis in patients who require such treatment. In other embodiments, the present invention provides mARC1-targeted RNAi constructs for use in methods for treating, preventing, or reducing hepatic fibrosis in patients who require such treatment. In some embodiments, patients at risk of developing hepatic fibrosis or cirrhosis are diagnosed with NAFLD. In other embodiments, patients at risk of developing hepatic fibrosis or cirrhosis are diagnosed with NASH. In yet another embodiment, patients at risk of developing hepatic fibrosis or cirrhosis are diagnosed with alcoholic steatohepatitis. In yet another embodiment, patients at risk of developing hepatic fibrosis or cirrhosis are diagnosed with hepatitis. In certain embodiments, administration of the RNAi construct of the present invention prevents or delays the onset of cirrhosis in patients.

[0150] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Examples]

[0151] Example 1. Inhibition of mARC1 expression in Ob / Ob animals modulates lipid levels. Genetic studies have reported an association between the A165T missense mutation in the MARC1 gene and reduced serum low-density lipoprotein (LDL) cholesterol and total cholesterol levels (Spracklen et al., Hum Mol Genet., Vol.26(9):1770-178, 2017; Emdin et al., bioRxiv 594523; / / doi.org / 10.1101 / 594523, 2019; and Emdin et al., PLoS Genet, Vol.16(4):e1008629, 2020). More recently, this mutation and other loss-of-function variants in the MARC1 gene have been associated with low levels of hepatic fat, reduced liver enzyme levels, and a reduced risk of cirrhosis (Emdin et al., 2019 and Emdin et al., 2020). To evaluate whether inhibition of mARC1 expression can reduce serum cholesterol levels, as observed in human carriers of the MARC1 A165T variant allele, aged obese mice (ob / ob) were administered siRNA molecules targeting the mouse Marc1 gene or control siRNA molecules. Because ob / ob mice are obese and have high lipid levels, they are often used as a model for type II diabetes and other metabolic disorders.

[0152] Male ob / ob animals (The Jackson Laboratory) aged 18-20 weeks were fed a standard solid diet (Harlan, 2020×Teklad global soy protein-free extruded rodent diet). For 6 weeks, the mice were administered subcutaneously every two weeks at a dose of 3 mg per kg of body weight, using only 0.2 ml of buffer solution (phosphate-buffered saline) (n=8), either mARC1-targeted siRNA (double-stranded number D-1000; n=8) or control siRNA (double-stranded number D-1002; n=8). As described in Example 2 below, siRNA molecules were synthesized and conjugated to a trivalent GalNAc moiety (structure shown in formula VII below). The structures of the respective siRNA molecules are shown in Tables 1 and 2 below. The animals were fasted and captured in week 6 for further analysis. Total RNA from captured animal livers was processed for qPCR analysis, and serum parameters were measured using a clinical analyzer (AU400 Chemistry Analyzer, Olympus). mRNA levels were first normalized to the 18S ribosomal RNA level in each liver sample, and then compared to the expression level of the buffer-only group. Data are presented as relative multiples of expression in the buffer-only group. Liver tissue was homogenized and extracted with isopropanol to measure total cholesterol and total triglycerides (ThermoFisher, Infinity Cholesterol Reagent and Infinity Triglyceride Reagent). All animal housing conditions and research protocols were approved by the Amgen Institutional Animal Care and Use Committee (IACUC). Mice were housed in a sterile AAALAC internationally accredited facility in a ventilated micro-isolator. The procedure and housing room were kept under positive pressure and regulated on a 12:12 dark:light cycle. All animals were freely supplied with reverse osmosis purified water via an automated watering system.

[0153] Animals treated with mARC1-targeted siRNA showed approximately 80% reduced hepatic mARC1 expression compared to animals administered only buffer (Figure 2A). Since hepatic expression of mARC2 mRNA was unaffected, the reduction in mARC1 expression by the siRNA molecule was specific (Figure 2B). Treatment with mARC1-targeted siRNA reduced serum high-density lipoprotein (HDL), LDL, and total cholesterol levels, as well as serum levels of alanine aminotransferase (ALT) and C-reactive protein (CRP) (Figures 3A-3H). Triglyceride levels in the liver were also reduced in ob / ob animals treated with mARC1-targeted siRNA (Figures 4A and 4B). In this animal model, hepatic expression of fibrosis genes in animals treated with mARC1-targeted siRNA did not change significantly compared to animals administered only buffer (data not shown).

[0154] The results of this series of experiments demonstrate that specific inhibition of mARC1 expression in the liver with mARC1-targeted siRNA molecules reduces serum cholesterol, LDL cholesterol, ALT levels, and liver triglycerides, demonstrating the causal effect of mARC1 in lipid regulation in hepatocytes. The observed reductions in serum cholesterol, LDL cholesterol, and ALT levels in ob / ob animals treated with mARC1-targeted siRNA are consistent with the reductions in the levels of these analytes observed in human carriers of the MARC1 A165T variant allele. Thus, inhibiting mARC1 expression with siRNA molecules such as those described herein may be useful in reducing cholesterol and triglyceride levels in patients with hypercholesterolemia or hyperlipidemia, and may also be therapeutic for other liver diseases such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic fatty liver disease, alcoholic steatohepatitis, hepatic fibrosis, and cirrhosis.

[0155] Example 2. Design and synthesis of the mARC1 siRNA molecule Using bioinformatics analysis of human MARC1 transcripts, candidate sequences for the design of therapeutic siRNA molecules targeting the human MARC1 gene were identified and are presented herein as Sequence ID No. 1 (Ensembl transcript number ENST00000366910.9; see Figure 1). The sequences were analyzed using an in-house siRNA design algorithm and selected if they met certain criteria. Bioinformatics analysis was performed in two stages. In the first stage, we evaluated various functions of the sequence, including cross-reactivity with MARC1 transcripts from cynomolgus monkeys (Macaca fascicularis; NCBI reference sequences XR_001490722.1, XR_001490723.1, XR_001490726.1, XR_273285.2, XM_005540901.2, XR_273286.2, XM_005540898.2 and XM_005540899.2), sequence identity with other human, cynomolgus monkey, and rodent gene sequences, and duplication with known human single nucleotide polymorphisms. In the second stage, the selection criteria were adjusted to predict off-target effects by evaluating sequences for seed region matching to human microRNA (miRNA) sequences, including sequences specific only to human MARC1 transcripts. Based on the results of bioinformatics analysis, 665 sequences were selected for initial synthesis and in vitro testing.

[0156] RNAi constructs were synthesized using solid-phase phosphoramidite chemistry. Synthesis was performed using MerMade12 or MerMade192X (Bioautomation) instruments. Various chemical modifications, including 2'-fluoromodified nucleotides, 2'-O-methylmodified nucleotides, inverted debased nucleotides, and phosphorothioate nucleotide internucleotide bonds, were incorporated into the molecules. RNAi constructs generally formalize into 19-21 base pair double helixes when annealed in a state without overhangs at the 3' end of the antisense and / or sense strands (either as a double-stranded bluntomer or with one or two overhangs of 2 nucleotides). For in vivo studies, the sense strand of the RNAi construct was conjugated to a trivalent N-acetyl-galactosamine (GalNAc) moiety, which is described in more detail below.

[0157] material Acetonitrile (DNA synthesis grade, AXO152-2505, EMD) Capping reagent A (80:10:10 (v / v / v) tetrahydrofuran / lutidine / acetic anhydride, BIO221 / 4000, EMD) Capping reagent B (16% 1-methylimidazole / tetrahydrofuran, BIO345 / 4000, EMD) Activating agent solution (0.25 M 5-(ethylthio)-1H-tetrazol (ETT) in acetonitrile, BIO152 / 0960, EMD) Detritylation reagent (3% dichloroacetic acid in dichloromethane, BIO830 / 4000, EMD) Oxidizing reagent (70:20:10 (v / v / v) tetrahydrofuran / pyridine / 0.02 M iodine in water, BIO420 / 4000, EMD) Diethylamine solution (20% DEA in acetonitrile, NC0017-0505, EMD) Thiolation reagent (0.05 M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4-dithiazol-3-thione in pyridine (BIOSULII / 160K)) 0.10 M adenosine, guanosine, and cytosine 5'-aminohexyl linker phosphoramidites, as well as 2'-methoxy and 2'-fluorophosphoramidites, in acetonitrile on a Molecular Trap Pack (0.5 g per 30 mL, Bioautomation) (Thermo Fisher Scientific) 0.10 M 2'-methoxyuridine phosphoramidite in 90:10 (v / v) acetonitrile / DMF on Molecular Trap Pack (0.5 g per 30 mL, Bioautomation) (Thermo Fisher Scientific) 0.10 M 2'-deoxy-reverse debase phosphoramidite (ChemGenes) in acetonitrile on a Molecular Trap Pack (0.5 g per 30 mL, Bioautomation) CPG support (high-load universal support, 500A (BH5-3500-G1), 79.6 μmol / g, 0.126 g (10 μmol)) or 1 μmol universal synthesis column, 500A, pipette-type body (MM5-3500-1, Bioautomation) Ammonium hydroxide (high concentration, JTBaker)

[0158] synthesis The reagent solution, phosphoramidite solution, and solvent were connected to a MerMade12 or MerMade192X instrument. Solid supports were added to each column (4 mL SPE tube with upper and lower frits for 10 μmol), and the columns were fixed to the instrument. The columns were washed twice with acetonitrile. The phosphoramidite and reagent solution lines were purged. Synthesis was started using Poseidon software. Synthesis was carried out by repeating the deprotection / coupling / oxidation / capping synthesis cycle. Specifically, the detritylation reagent was added to the solid support to remove the 5'-dimethoxytrityl (DMT) protecting group. The solid support was washed with acetonitrile. The phosphoramidite and activator solution were added to the support and subsequently incubated to bond the incoming nucleotides to the free 5'-hydroxyl groups. The support was washed with acetonitrile. The oxidizing or thiolation reagent was added to the support to convert the phosphite triester to phosphate triester or phosphorothioate. Capping reagents A and B were added to the support to terminate any unreacted oligonucleotide chains. The support was washed with acetonitrile. After the final reaction cycle, the resin was washed with diethylamine solution to remove the 2-cyanoethyl protecting group. The support was washed with acetonitrile and dried under vacuum.

[0159] GalNAc conjugation A sense chain for conjugation to the trivalent GalNAc moiety (structure shown in formula VII below) was prepared using a 5'-aminohexyl linker. After automated synthesis, the column was removed from the instrument and transferred to a vacuum manifold in a hood. The 5'-monomethoxytrityl (MMT) protecting group was removed from the solid support by vacuum filtration with 2 mL aliquots of 1% trifluoroacetic acid (TFA) in dichloromethane (DCM). Once no further orange / yellow coloration was observed in the eluate, the resin was washed with dichloromethane. The resin was then washed with 10% diisopropylethylamine in 5 mL of N,N-dimethylformamide (DMF). In a separate vial, a solution of GalNAc3-Lys2-Ahx (67 mg, 40 μmol) in DMF (0.5 mL) (its structure and synthesis are described below) was prepared using 1,1,3,3-tetramethyluronium tetrafluoroborate (TATU, 12.83 mg, 40 μmol) and diisopropylethylamine (DIEA, 13.9 μL, 80 μmol). The activated coupling solution was added to the resin, the column was capped, and incubated overnight at room temperature. The resin was washed with DMF and DCM and dried under vacuum.

[0160] Cutting The synthesis column was removed from the synthesis apparatus or vacuum manifold and transferred to the cutting apparatus. 4 × 1 mL (for 10 μmol) or 4 × 250 μL (for 1 μmol) of high-concentration ammonium hydroxide was added to the solid support. The eluate was collected into 24 or 96-well deep-well plates by gravity or reduced-pressure filtration, respectively. The plates were sealed and bolted into a cleavage chuck (Bioautomation), and the mixture was heated at 55°C for 4 hours. The plates were transferred to a freezer and cooled for 20 minutes before the cleavage chuck was opened in the hood.

[0161] Analysis and purification A portion of the cleavage solution was analyzed and purified by anion exchange chromatography. The pooled fraction was desalted by size exclusion chromatography and analyzed by ion-pair reverse-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pooled fraction was freeze-dried to obtain a white amorphous powder.

[0162] Analytical anion exchange chromatography (AEX): Column: Thermo DNAPac PA200RS (4.6 × 50 mm, 4 μm) Equipment: Agilent 1100 HPLC Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5 Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide Flow rate: 1 mL / min at 40°C Gradient: 20-65% B over 6.2 minutes

[0163] Preparative anion exchange chromatography (AEX): Column: Tosoh Corporation TSK Gel SuperQ-5PW, 21 x 150 mm, 13 μm Equipment: Agilent1200 HPLC Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5 Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide Flow rate: 8mL / min Injection volume: 5mL Gradient: Sense chain 35-55%B over 40 minutes, antisense chain 50-100%B over 40 minutes

[0164] Preparative size exclusion chromatography (SEC): Columns: 3×GE Hi-Prep 26 / 10, in series Equipment: GE AKTA Pure Buffer solution: 20% ethanol aqueous solution Flow rate: 10mL / min Injection volume: 45 mL using a sample load pump

[0165] Ion-pair reversed-phase (IP-RP) HPLC: Column: Water Xbridge BEH OST C18, 2.5 μm, 2.1 × 50 mm Equipment: Agilent 1100 HPLC Buffer A: 15.7 mM DIEA in water, 50 mM hexafluoroisopropanol (HFIP) Buffer B: 15.7 mM DIEA and 50 mM HFIP in 50:50 water / acetonitrile Flow rate: 0.5mL / min Gradient: 10-30% over 6 minutes

[0166] annealing Small amounts of sense and antisense strands were weighed into individual vials. Phosphate-buffered saline (PBS, Gibco) was added to the vials to a concentration of approximately 2 mM based on dry weight. The actual sample concentration was measured on NanoDrop One (ssDNA, extinction coefficient = 33 μg / OD260). Next, the two strands were mixed in equimolar ratios, and the sample was heated in a 90°C incubator for 5 minutes and slowly cooled to room temperature. The sample was analyzed by AEX. The double helix was registered and subjected to in vitro and in vivo testing as described in more detail in Examples 3 and 4 below.

[0167] Preparation of GalNAc3-Lys2-Ahx [ka] In the formula, X = O or S. The wavy line indicates the binding site to the 5' terminal nucleotide of the sense strand of the RNAi construct. The GalNAc moiety was bound to the 5' carbon of the 5' terminal nucleotide of the sense strand, except when the inverted debasing (invAb) deoxyribonucleotide was the 5' terminal nucleotide and bound to an adjacent nucleotide via a 5'-5' nucleotide bond. In this case, the GalNAc moiety was bound to the 3' carbon of the inverted debasing deoxyribonucleotide.

[0168] Fmoc-Ahx-OH (1.13 g, 3.19 mmol) was added to 30 mL of DCM in a 50 mL Falcon tube, followed by DIEA (2.23 mL, 12.78 mmol). The solution was added to 2-Cl trityl chloride resin (3.03 g, 4.79 mmol) in a 50 mL centrifuge tube and immersed in a shaker for 2 hours. The solvent was drained, and the resin was washed with 17:2:1 DCM / MeOH / DIEA (30 mL x 2) and DCM (30 mL x 4) and then dried. UV spectrophotometric detection at 290 nm determined the amount added to be 0.76 mmol / g.

[0169] 3 g of 2-Cl trityl resin was suspended in 20 mL of DMF with 20% 4-methylpiperidine, and the solvent was drained after 30 minutes. This process was repeated once more, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0170] To a solution of Fmoc-Lys(ivDde)-OH (3.45 g, 6 mmol) in DMF (20 mL), TATU (1.94 g, 6 mmol) was added, followed by DIEA (1.83 mL, 10.5 mmol). The solution was then added to the deprotected resin, and the suspension was left on a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0171] The resin was treated with 20% 4-methylpiperidine in DMF (15 mL), and the solvent was drained after 10 minutes. This process was repeated once more, and the resin was washed with DMF (15 mL x 4) and DCM (15 mL x 4).

[0172] To a solution of Fmoc-Lys(Fmoc)-OH (3.54 g, 6 mmol) in DMF (20 mL), TATU (1.94 g, 6 mmol) was added, followed by DIEA (1.83 mL, 10.5 mmol). The solution was then added to the deprotected resin, and the suspension was left on a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0173] The resin was treated with 5% hydrazine in 20 mL of DMF, and the solvent was drained after 5 minutes. This process was repeated four more times, and the resin was washed with 30 mL x 4 of DMF and 30 mL x 4 of DCM.

[0174] To a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (4.47 g, 10 mmol) in DMF (40 mL), TATU (3.22 g, 10 mmol) was added, and the solution was stirred for 5 minutes. DIEA (2.96 mL, 17 mmol) was added to this solution, and the mixture was then added to the resin. The suspension was kept at room temperature overnight, and the solvent was drained. The resin was washed with DMF (3 × 30 mL) and DCM (3 × 30 mL).

[0175] The resin was treated with 1% TFA in DCM (30 mL, containing 3% triisopropylsilane), and the solvent was drained after 5 minutes. This process was repeated three more times, and the combined filtrate was concentrated under reduced pressure. The residue was pulverized with diethyl ether (50 mL), the suspension was filtered, and the product was dried to obtain the crude product. The crude product was purified by reverse-phase chromatography and eluted with 0-20% MeCN in water. The fractions were combined and freeze-dried to obtain the product as a white solid.

[0176] Table 1 below lists the unmodified sense and antisense sequences of molecules prioritized from bioinformatics analysis. The range of nucleotides targeted by the siRNA molecules within each sequence family in the human MARC1 transcript (SEQ ID NO: 1) is also shown in Table 1. Double-strand numbers D-1000 to D-1003 are designed to target the Marc1 mouse transcript and do not cross-react with the human MARC1 transcript. Table 2 provides the sense and antisense sequences, including chemical modifications. Based on the activity in in vitro cell-based assays and in vivo mouse studies described in Examples 3 and 4, sequences targeting specific regions of the human MARC1 transcript were selected for structure-activity relationship (SAR) testing. Nucleotide sequences are listed according to the following notation: a, u, g, and c = corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro")ribonucleotides; and invAb = inverted debased deoxynucleotide (i.e., a debased deoxynucleotide that, when located at the 3' end of a chain, is linked to an adjacent nucleotide via its 3' substituent (3'-3' linkage) or, when located at the 5' end of a chain, is linked to an adjacent nucleotide via its 5' substituent (5'-5' internucleotide linkage)). The insertion of "s" in a sequence indicates that two adjacent nucleotides are linked by a phosphorothiodiester group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise indicated, all other nucleotides are linked by a 3'-5' phosphodiester group. [GalNAc3] represents the GalNAc moiety shown in formula VII, and when "s" follows the [GalNAc3] notation, it is covalently bonded to the 5' terminal nucleotide at the 5' end of the sense strand via a phosphodiester bond or a phosphorothioate bond. If the invAb nucleotide was the 5' terminal nucleotide at the 5' end of the sense strand, it would be linked to an adjacent nucleotide via a 5'-5' linkage, and the GalNAc moiety would be covalently bonded to the 3' carbon of the invAb nucleotide. On the other hand, the GalNAc moiety would be covalently bonded to the 5' carbon of the 5' terminal nucleotide of the sense strand.

[0177] Table 1

[0178] Table 2

[0179] Table 3

[0180] Table 4

[0181] Table 5

[0182] Table 6

[0183] Table 7

[0184] Table 8

[0185] Table 9

[0186] Table 10

[0187] Table 11

[0188] Table 12

[0189] Table 13

[0190] Table 14

[0191] Table 15

[0192] Table 16

[0193] Table 17

[0194] Table 18

[0195] Table 19

[0196] Table 20

[0197] Table 21

[0198] Table 22

[0199] Table 23

[0200] Table 24

[0201] Table 25

[0202] Table 26

[0203] Table 27

[0204] Table 28

[0205] Table 29

[0206] Table 30

[0207] Table 31

[0208] Table 32

[0209] Table 33

[0210] Table 34

[0211] Table 35

[0212] Table 36

[0213] Table 37

[0214] Table 38

[0215] Table 39

[0216] Table 40

[0217] Table 41

[0218] Table 42

[0219] [Table 43]

[0220] [Table 44]

[0221] [Table 45]

[0222] [Table 46]

[0223] [Table 47]

[0224] [Table 48]

[0225] [Table 49]

[0226] [Table 50]

[0227] [Table 51]

[0228] Example 3. In vitro evaluation of the mARC1 siRNA molecule in a cell-based assay. mARC1 siRNA molecules with various sequences prioritized from the bioinformatics analysis described in Example 2 were screened for their effectiveness in reducing human mARC1 mRNA using RNA FISH (fluorescence in situ hybridization) assay. Hep3B cells (purchased from ATCC) were cultured in Eagle's Minimal Essential Medium (EMEM) (ATCC 30-2003) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning), and siRNA was transfected into the cells by reverse transfection using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific). The mARC1 siRNA molecules were tested in a 10-point dose-response format, 3-fold dilution, in the range of 500 nM to 25 pM (test 1), 25 nM to 1 pM (test 2), or 100 nM to 5 pM (test 3), at final concentrations. 1 μL of test siRNA molecule or phosphate-buffered saline (PBS) vehicle and 4 μL of base EMEM (unsupplemented) were added to a CellCarrier-384 Ultra assay plate (PerkinElmer) PDL-coated using a Bravo automated liquid processing platform (Agilent). Subsequently, 5 μL of pre-diluted Lipofectamine RNAiMAX (Thermo Fisher Scientific) (0.035 μL of RNAiMAX in 5 μL of EMEM) was dispensed into the assay plate using a Multidrop Combi reagent dispenser (Thermo Fisher Scientific) into the unsupplemented base EMEM. After incubating the siRNA / RNAiMAX mixture at room temperature (RT) for 20 minutes, 30 μL of Hep3B cells (2000 cells per well) in EMEM supplemented with 10% FBS and 1% PS were added to the transfection complex using a Multidrop Combi reagent dispenser. The assay plate was incubated at RT for 20 minutes before being placed in an incubator. The cells were incubated at 37°C and 5% CO2 for 72 hours.RNA FISH assays were performed 72 hours after siRNA transfection on an in-house assembled automated FISH assay platform using the manufacturer's assay reagents and protocol (QuantiGene® ViewRNA HC Screening Assay from Thermo Fisher Scientific). In short, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) at RT for 15 minutes, permeabilized with a surfactant at RT for 3 minutes, and then treated with a protease solution at RT for 10 minutes. Target-specific probes (Thermo Fisher Scientific) or a vehicle (target probe diluent without the target probe as a negative control) were incubated for 3 hours, while the preamplifier, amplifier, and labeled probe were incubated for 1 hour each. All hybridization steps were performed at 40°C in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific). After hybridization, cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 minutes and then imaged using the Opera Phenix high-content screening system (PerkinElmer). The images were analyzed using the Columbus image data storage and analysis system (PerkinElmer) to obtain the average number of spots per cell. The average number of spots per cell was normalized using high (PBS containing the target probe) and low (PBS without the target probe) control wells. The normalized values ​​were plotted against the total siRNA concentration, and the data were fitted to a four-parameter sigmoid model using Genedata Screener data analysis software (Genedata) to obtain IC50 and maximum activity values. If the data could not be fitted to the model, the IC50 value was not calculated, and only the maximum activity value was reported.

[0229] First, the mARC1 siRNA molecule was screened at 10 different concentrations ranging from 500 nM to 25 pM in the first assay. siRNA molecules that showed significant activity in the first assay were screened again in the second and third assays at 10 different concentrations within a narrower concentration range (Assemble 2: 25 nM to 1 pM; Assemble 3: 100 nM to 5 pM). The results of all three assays are shown in Table 3 below.

[0230] [Table 52]

[0231] [Table 53]

[0232] [Table 54]

[0233] [Table 55]

[0234] [Table 56]

[0235] [Table 57]

[0236] Of the first 257 mARC1 siRNA molecules evaluated by RNA FISH assay, 74 molecules showed an average knockdown of 80% or more of human mARC1 mRNA in assays 2 and 3, and had IC50 values ​​in the nanomolar range of at least one order of magnitude. In particular, 32 molecules (double-strand numbers D-1092; D-1093; D-1139; D-1061; D-1138; D-1095; D-1191; D-1180; D-1090; D-1062; D-1177; D-1083; D-1245; D-1067; D-1143; D-1170; D-1044) D-1096;D-1113;D-1086;D-1256;D-1189;D-1091;D-1174;D-1185;D-1066;D-1171;D-1140;D-1130;D-1068;D-1243;D-1074) reduced human mARC1 mRNA by at least 85% in one or both of assays 2 and 3.

[0237] In a second series of experiments, additional mARC1 siRNA molecules were evaluated in RNA FISH assays at 10 different concentrations ranging from 100 nM to 5 pM, and the IC50 and maximum activity values ​​were calculated as described above. The assay results from this second series of experiments are shown in Table 4 below. The assays were repeated for subsets of molecules. For these molecules, the IC50 and maximum activity values ​​for both implementations are shown.

[0238] [Table 58]

[0239] [Table 59]

[0240] [Table 60]

[0241] [Table 61]

[0242] Table 62

[0243] Table 63

[0244] Table 64

[0245] Table 65

[0246] Table 66

[0247] Table 67

[0248] Table 68

[0249] Table 69

[0250] Of the 406 additional mARC1 siRNA molecules that target different regions of the human mARC1 transcript, 128 molecules reduced human mARC1 mRNA by more than 85% in Hep3B cells. 46 of these molecules (double-strand numbers D-1061; D-1093; D-1220; D-1276; D-1284; D-1298; D-1310; D-1311; D-1338; D-1363; D-1367; D-1375; D-1381; D-1382; D-1383; D-1386; D-1387; D-1388; D-1389; D-1390; D-1396; D-1400; D-1401) D-1402;D-1405;D-1407;D-1416;D-1420;D-1421;D-1441;D-1451;D-1487;D-1489;D-1491;D-1503;D-1504;D-1515;D-1549;D-1576;D-1581;D-1595;D-1596;D-1606;D-1626;D-1633;and D-1662) reduced human mARC1 mRNA by at least 90%, and the majority of the molecules had an IC50 value of less than 1 nM.

[0251] Example 4. In vivo efficacy of siRNA molecules in an AAV human mARC1 mouse model. To evaluate the efficacy of the mARC1 siRNA molecule in vivo, the sense strand in each siRNA molecule was conjugated to the trivalent GalNAc portion shown in formula VII by the method described in Example 2, and the mARC1 siRNA molecule was administered to mice expressing the human MARC1 gene. 10-12 week old c57BL / 6 mice (The Jackson Laboratory) were fed a standard solid diet (Harlan, 2020×Teklad global soy protein-free extruded rodent diet). The mice were given 1 × 10⁶ doses of adeno-associated virus (AAV) encoding the human MARC1 gene (AAV-hmARC1) per animal. 11The AAV-hmARC1 was administered intraperitoneally (ip) at the dose of a genome copy (GC). One week after AAV-hmARC1 injection, mice were given a single subcutaneous (sc) injection of mARC1 siRNA molecules in buffer or in buffer at doses of 0.5 mg, 1 mg, or 3 mg per kg of body weight (n=3 in each group). The animals were fasted and captured four weeks after siRNA administration for further analysis. Total RNA from the livers of the captured animals was processed for qPCR analysis, and serum parameters were measured using a clinical analyzer (AU400 Chemistry Analyzer, Olympus). The percentage change in human mARC1 mRNA in the liver of each animal was calculated by comparing it with the human mARC1 mRNA liver levels in control animals expressing human mARC1 mRNA and administered only buffer (i.e., AAV-hmARC1-only animals).

[0252] The most effective mARC1 siRNA molecule from the in vitro activity assay described in Example 3 was evaluated for in vivo efficacy in this model. To further improve in vivo efficacy and tolerance by altering the chemical modification pattern, mARC1 siRNA molecules showing significant in vivo knockdown activity were further evaluated in SAR studies. The results of 18 individual studies in AAV-hmARC1 mouse models with different mARC1 siRNA molecules are shown in Tables 5-22 below. Data are expressed as the mean percentage change from control at week 5 of each treatment group study (4 weeks after siRNA injection) (n=3 animals / group). When two mARC1 siRNA molecules have the same trigger family designation as another mARC1 siRNA molecule, the two molecules have the same core sequence (i.e., target the same region of the mARC1 transcript), but their chemical modification patterns differ.

[0253] [Table 70]

[0254] [Table 71]

[0255] Table 72

[0256] Table 73

[0257] Table 74

[0258] Table 75

[0259] Table 76

[0260] Table 77

[0261] Table 78

[0262] Table 79

[0263] Table 80

[0264] Table 81

[0265] Table 82

[0266] Table 83

[0267] Table 84

[0268] Table 85

[0269] Table 86

[0270] Table 87

[0271] Two mARC1 siRNA molecules (double-strand numbers D-2042 and D-2081) that showed significant silencing activity in early in vivo studies were used as benchmark compounds in later in vivo studies. Seventy different mARC1 siRNA molecules reduced human mARC1 mRNA by more than 75% four weeks after a single sc injection at a dose of 1 mg / kg in AAV-hmARC1 mice. Some of the tested mARC1 siRNA molecules, including D-2081, D-2241, D-2255, and D-2258, were particularly potent, as evidenced by the more than 85% reduction in human mARC1 mRNA four weeks after a single sc injection of just 0.5 mg / kg. Furthermore, mARC1 siRNA molecules targeting specific regions of the human mARC1 transcript were observed to reduce human mARC1 mRNA even more significantly in vivo compared to mARC1 siRNA molecules targeting other regions of the transcript. For example, mARC1 siRNA molecules containing antisense strands with sequences complementary to the human mARC1 transcript region (SEQ ID NO: 1) at nucleotides 1205–1250, 1345–1375, or 2039–2078 showed significant knockdown activity 4 weeks after a single 1 mg / kg sc injection (Table 23). Table 23 summarizes the mean percentage change in human mARC1 mRNA liver levels from the above studies for siRNA molecules having the same chemical modification pattern and targeting human transcripts in specified nucleotide ranges. mARC1 siRNA molecules targeting human transcripts at nucleotides 1211–1236 were particularly effective, as a single sc dose of 1 mg / kg of such siRNA molecules resulted in a more than 80% reduction in human mARC1 mRNA levels at least 4 weeks after administration.

[0272] [Table 88]

[0273] [Table 89]

[0274] [Table 90]

[0275] Example 5. Efficacy of mARC1 siRNA in the treatment of NASH in a mouse model. To determine whether inhibiting mARC1 expression could be therapeutic for fatty liver disease, mice fed a 0.2% cholesterol diet (TD190883 diet) were administered either an siRNA molecule targeting the mouse Marc1 gene or a control siRNA molecule. The TD190883 diet contains 0.2% cholesterol, 20% fructose, 12% sucrose, and 22% hydrogenated vegetable oil (HVO). Similar diets have been shown to induce NAFLD and NASH characteristics in mice fed this diet for several weeks (see, e.g., Zhong et al., Digestion, Vol.101:522-535, 2020 and Kroh et al., Gastroenterol Res Pract. Vol.2020:7347068, 2020, doi:10.1155 / 2020 / 7347068).

[0276] Six-week-old male c57BL / 6 mice (Charles River Laboratories) were fed either a standard solid diet (Harlan, 2020×Teklad global soy protein-free extruded rodent diet) or a 0.2% cholesterol diet (TD190883, Envigo). Mice fed the 0.2% cholesterol diet were administered subcutaneously every two weeks for 24 weeks, using only 0.2 ml of buffer solution (phosphate-buffered saline), either mARC1-targeted siRNA (double-stranded number D-1000) or control siRNA (double-stranded number D-1002), at a dose of 3 mg per kg of body weight. As described in Example 2, siRNA molecules were synthesized and conjugated to the trivalent GalNAc moiety (structure shown in formula VII below). The structures of the respective siRNA molecules are shown in Tables 1 and 2. The animals were fasted and captured at week 24 for further analysis. Total RNA from captured animal livers was processed for qPCR analysis, and serum parameters were measured using a clinical analyzer (AU400 Chemistry Analyzer, Olympus). mRNA levels were first normalized to the 18S ribosomal RNA level in each liver sample, and then compared to the expression level of the solid diet control group. Data are presented as relative multiples of expression in the solid diet control group. Liver tissue was homogenized and extracted with isopropanol to measure total cholesterol and total triglycerides (ThermoFisher, Infinity Cholesterol and Infinity Triglycerides). All animal housing conditions and research protocols were approved by the Amgen Institutional Animal Care and Use Committee (IACUC). Mice were housed in a sterile AAALAC internationally accredited facility in a ventilated micro-isolator. The procedure and housing room were kept under positive pressure and regulated on a 12:12 dark:light cycle. All animals were freely supplied with reverse osmosis purified water via an automated watering system.

[0277] In mice fed a 0.2% cholesterol diet, hepatic expression of both mARC1 and mARC2 was reduced. In animals treated with mARC1-targeted siRNA, mARC1 expression was further reduced, but mARC2 expression was not further reduced (Figures 5A and 5B). As expected, mice fed a 0.2% cholesterol diet showed increased serum levels of liver enzymes (AST and ALT), cholesterol, LDL cholesterol (LDL-C), and HDL cholesterol (HDL-C) during the study (Figures 6A-6E). Treatment with mARC1-targeted siRNA reduced diet-induced increases in serum cholesterol, LDL-C, and HDL-C (Figures 6C-6E). mARC1 siRNA treatment also tended to reduce diet-induced serum levels of liver enzymes (Figures 6A-6B). Animals fed a 0.2% cholesterol diet showed increased body weight and liver weight after 24 weeks (Figures 7A and 7B). In animals fed a 0.2% cholesterol diet, liver triglyceride and cholesterol levels also increased after 24 weeks (Figures 7C and 7D). mARC1 siRNA treatment did not significantly reduce diet-induced increases in body weight, liver weight, liver triglyceride levels, or liver cholesterol levels (Figures 7A-7D).

[0278] In summary, the results of this study show that inhibiting mARC1 liver expression with a mARC1-targeted siRNA molecule reduces serum cholesterol, LDL-C, HDL-C, and liver enzymes in a mouse model of NASH, suggesting that the mARC1 siRNA molecule could be a novel therapeutic approach for treating this disease and other fatty liver disorders.

[0279] Example 6. Effect of mismatch on the potency of the mRNA1 siRNA molecule To evaluate the effect of base pair mismatch on the potency of the mARC1 siRNA molecule, analogues of the most potent subset of siRNA molecules were synthesized with different nucleotides at positions 6 or 8 from the 5' end of the antisense strand, so that a base pair mismatch would be generated at that position when the antisense strand hybridized to its target region of the mARC1 mRNA transcript. However, in each analogue, the sense strand sequence was designed to be perfectly complementary to the antisense strand sequence, so no mismatch was generated between the sense and antisense strands in the siRNA duplex. The unmodified and modified sequences of each mismatch analog (double-strand numbers D-2514 to D-2561) and the parent siRNA molecules (double-strand numbers D-2052, D-2072, D-2076, D-2077, D-2079, D-2081, D-2105, D-2108, D-2111, D-2113, D-2115, D-2118, D-2142, D-2136, D-2189, D-2196, D-2238, D-2241, D-2254, D-2258, D-2301, D-2462, D-2465, and D-2510) are provided in Tables 1 and 2, respectively. The efficacy of mismatch analogs and parental siRNA molecules in reducing human mARC1 mRNA levels was evaluated in Hep3B cells using the in vitro RNA FISH assay described in Example 3 above. Ten different concentrations of each siRNA molecule, ranging from 100 nM to 5 pM, were tested, and the IC50 and maximum activity values ​​were calculated from the dose-response curves described in Example 3. The results of these assays are shown in Table 24 below.

[0280] [Table 91]

[0281] [Table 92]

[0282] [Table 93]

[0283] In most molecules, mismatches at position 6 or 8 within the seed region of the antisense strand did not significantly affect the maximum knockdown activity or potency of the siRNA molecule compared to the parent molecule where the antisense strand was perfectly complementary to the target mARC1 mRNA sequence. These results are somewhat surprising, given that the seed region of the antisense strand (i.e., nucleotides 2–8 from the 5' end) is considered important for on-target efficacy.

[0284] Example 7. In vivo efficacy of the mARC1 siRNA molecule in non-human primates. The efficacy and pharmacokinetic profiles of three different mARC1 siRNA molecules (double-stranded numbers D-2241, D-2081, or D-2258) were evaluated in cynomolgus monkeys. Each of the three different mARC1 siRNA molecules had an antisense strand sequence that cross-reacted with the MARC1 gene of cynomolgus monkeys (Macaca fascicularis). Treatment-naive female cynomolgus monkeys born in Mauritius, aged 22–48 months, were obtained from Charles River Laboratories, Inc., Research Model Services (Houston, TX). A single 3 mg / kg subcutaneous (sc) injection of one of the GalNAc-conjugated mARC1 siRNA molecules (double-stranded numbers D-2241, D-2081, or D-2258) in 1× phosphate-buffered saline was administered to the scapula and mid-back of the animals (n=3 / treatment group). Serum was prepared from whole blood collected at the following time points after administration: 0.083, 0.25, 1, 2, 4, 24, 28, 96, 168, 264, 336, 456, 528, 576, 720, 864, and 1056 hours. Surgical liver biopsies (approximately 100 mg of tissue per liver lobe, left and right) were taken under anesthesia before the procedure (either 13 or 7 days prior) and on 14 and 30 days after administration. Liver samples were dissected and collected on 44 days after administration.

[0285] Serum and hepatic pharmacokinetics To determine the serum and liver pharmacokinetic profiles of each GalNAc-conjugate mARC1 siRNA molecule, serum and liver samples collected at various time points after treatment with a single 3 mg / kg sc dose of mARC1 siRNA molecule were analyzed for each mARC1 siRNA molecule (antisense and sense strands) using a plate-based oligonucleotide electrochemiluminescence (POE) immunoassay similar to that described in Thayer et al., Sci. Rep., Vol. 10(1): 10425, 2020. Oligonucleotide capture (biotin) and detection (digoxigenin) probes were custom synthesized from Qiagen Inc. (Hilden, Germany). Their sequences are listed in Table 25 below. Liver samples were homogenized in a lysis buffer containing 50 mM Tris HCl, 100 nM NaCl, 0.1% Triton X100, and a Roche protease inhibitor cocktail (11836170001) until the final concentration reached 200 mg / mL. For bioanalysis, GalNAc-mARC1 siRNA standards were spiked into serum or liver homogenates over a concentration range of 0.13–2500 ng / mL. Subsequently, the standards and biological samples were diluted 1:10 in a 96-well PCR plate to a final volume of 50 μL. Oligonucleotide capture and detection probes were prepared in a hybridization buffer consisting of 60 mM Na2PO4 (pH 7.0, dibasic), 1 M NaCl, 5 mM EDTA, and 0.02% Tween 20. The probe was combined and added to the PCR plate at a final concentration of 10 nM, resulting in a total sample volume of 100 μL per well. Hybridization was performed using a thermal cycler under the following conditions: 90°C for 5 minutes, 40°C for 30 minutes, and final retention at 12°C. After hybridization, 45 μL of the sample was transferred to a Meso Scale Diagnostics LLC MSD Gold 96-well Streptavidin SECTOR plate (L15SA) and incubated at room temperature for 30 minutes with shaking. The plate was washed with SerCare Life Sciences 1X KPL immunoassay washing solution (5150-0011).After washing, the plates were incubated for 1 hour with 50 μL of 0.5 μg / mL ruthenium-labeled anti-digoxigenin antibody diluted in ThermoFisher Scientific SuperBlock T20 TBS blocking buffer (37536). After a final wash, 150 μL of Meso Scale Diagnostics LLC 1X MSD Read buffer T (R92TC) was added and read on a Meso Scale Diagnostics LLC Meso Sector S 600 instrument. Serum and liver concentrations of the mARC1 siRNA molecule were interpolated from standard curves using a 4-parameter logistic model and a 1 / Y2 weighting coefficient within Watson LIMS biological analysis software version 7.5 (ThermoFisher Scientific). Liver concentrations were converted from ng / mL to ng / mg by dividing by 200 mg / mL. Serum pharmacokinetic parameters were determined 0.083 to 24 hours after administration using non-compartmental analysis within Phoenix WinNonlin software version 8.3.2.116 (Pharsight).

[0286] [Table 94]

[0287] The serum concentration-time profiles of antisense and sense strand concentrations for each of the three different mARC1 siRNA molecules are shown in Figures 8A-8F. As summarized in Table 26, the mean maximum observed antisense strand concentration in serum (C) max The concentrations were 511, 496, and 321 ng / mL for D-2241, D-2258, and D-2081 2.0 to 4.0 hours after administration. The mean area under the concentration-time curve (AUC) of serum antisense chains from dose initiation to 24 hours post-administration. 0~24時間The values ​​were 6399, 5040, and 4137 h*ng / mL for D-2258, D-2241, and D-2081, respectively. The ratio of serum concentrations of the sense strand to the antisense strand of double helix number D-2258 indicates potential instability of the double helix with strand separation, which may occur at the injection site or in systemic circulation. Liver concentrations of antisense and sense strand siRNAs at 14, 30, and 44 days post-administration are reported in Table 27. Liver antisense strand concentrations at 14 days were highest for double helix number D-2081, followed by D-2241, and then D-2258. Consistent with the serum pharmacokinetic profile, the ratio of liver concentrations of the sense strand to the antisense strand of double helix number D-2258 indicates strand separation.

[0288] [Table 95]

[0289] [Table 96]

[0290] Liver mARC1 mRNA silencing Three GalNAc-conjugate mARC1 siRNA molecules (double-strand numbers D-2241, D-2081, and D-2258) were evaluated for their efficacy in knocking down mARC1 mRNA levels after a 3 mg / kg sc dose in cynomolgus monkey liver. RNA was purified from liver rapidly frozen using the ThermoFisher Scientific MagMAX-96 Total RNA Isolation Kit (AM1830), and sample integrity (260 / 280 ratio) and RNA concentration were determined using a ThermoFisher Scientific NanoDrop 2000 spectrophotometer (ND-2000). One-step reverse transcription polymerase chain reaction (RT-PCR) was performed using the ThermoFisher Scientific TaqMan® RNA-to-CT 1-Step Kit (4392938). The reaction was combined in a 96-well PCR plate by mixing 50 ng of RNA template with 2× TaqMan RT-PCR Mix, 40× TaqMan RT Enzyme Mix, 20× mARC1 primers / probes (IDT, forward primer 5'-TTCAGGATGCGATGT CTATGC-3' (SEQ ID NO: 3671), reverse primer 5'-TGCCCAAAGAGTGGTGATTT-3' (SEQ ID NO: 3672), probe 5'- / 56-FAM / AGCCGCTGG (SEQ ID NO: 3673) / ZEN / AAACACT GAAGAGTT (SEQ ID NO: 3674) / 3IABkFQ / -3'), and 20× glyceraldehyde-3-phosphate dehydrogenase primer / probe (GAPDH; ThermoFisher Scientific, Mf04392546_g1 VIC-MGB). RT-PCR was performed using a ThermoFisher Scientific QuantStudio 7 Flex Real-Time PCR System (4485701) under the following conditions: 40 cycles of 30 minutes at 48°C and 10 minutes at 90°C, followed by 15 seconds at 90°C and 1 minute at 60°C. mRNA expression in each sample was normalized by taking the ratio of the concentration of the target gene (mARC1) over the concentration of the housekeeping gene (GAPDH).Next, the percentage of mARC1 mRNA expression (%) after siRNA administration (days 14, 30, and 44) ​​was calculated for each animal replication per treatment group compared to pre-treatment (days -13 or -7), and this was expressed as the pre-treatment residual percentage. Finally, the silencing percentage (%) of the mARC1 mRNA transcript was calculated by subtracting the pre-treatment residual percentage from 100%. Both the pre-treatment residual percentage and silencing percentage of mRNA are summarized in Table 28 below. Double-stranded number D-2241 was the most potent GalNAc-conjugate mARC1 siRNA molecule tested, which reduced mARC1 liver mRNA in cynomolgus monkeys to <20% residual (>80% silencing) on ​​days 14, 30, and 44 after a single subcutaneous injection.

[0291] [Table 97]

[0292] Liver mARC1 protein silencing The efficacy of three GalNAc-conjugate mARC1 siRNA molecules (double-strand numbers D-2241, D-2081, and D-2258) in knockdown of mARC1 protein levels after a 3 mg / kg sc dose in cynomolgus monkey liver was also evaluated. Rapidly frozen liver tissue was homogenized at 200 mg / mL in Boston Bioproduct NP-40 lysis buffer (BP-119) containing ThermoFisher Scientific protease inhibitor tablets (A32963). Subsequently, the homogenate was spun down at 10,000 × g for 10 minutes at 4°C, and the supernatant was transferred to a 2 mL 96-deep-well plate. The supernatant was incubated at room temperature for 15 minutes and treated with 1% trifluoroacetic acid in methanol with shaking at 1400 rpm. The precipitated protein was pelletized at 4,000 rpm for 15 minutes, the supernatant was aspirated, and the pellet was washed twice with methanol. The obtained protein was denatured by reduction in a solution containing 10 mM tris(2-carboxyethyl)phosphine (ThermoFisher Scientific, 77720) and 8 M urea at 37°C for 30 minutes. Subsequently, iodoacetamide (20 mM; ThermoFisher Scientific, A39271) was added to the sample in 20 mM ammonium bicarbonate buffer and incubated at room temperature for 30 minutes. 30 μg of trypsin (ThermoFisher Scientific, A90058) and 10 pmol of a stable isotope-labeled (SIL) peptide (custom peptide from ThermoFisher Scientific; [ka] The sample was digested overnight at 37°C with the addition of ) . The digestion reaction was stopped with 20% formic acid, and the sample was prepared for solid-phase extraction (SPE) desalting (Waters Corporation, 186008052). Before adding the sample, the SPE plate was conditioned with methanol and washed once with 1% acetonitrile. The sample was added to the conditioned SPE plate, and the analyte was eluted using 70% acetonitrile. The eluate was resuspended in 10 mM ammonium formate at pH 10 and injected into an Agilent 1260 Infinity Bio-inert Analytical-scale Fraction Collector (G5664A). The fractionated sample (11th fraction) was resuspended in 0.1% formic acid solution for analysis on a ThermoFisher Scientific Ultimate 3000 ultra-high-performance liquid chromatography (LC) system connected to an Orbitrap Lumos mass spectrometer (MS). The LC method was performed as follows: Trapping was performed at a column temperature of 45°C with 3% acetonitrile / water (8 μL / min) and an analytical gradient of 3.0–36% acetonitrile / water over 1.0–12.1 minutes (350 nL / min). Concurrent reaction monitoring experiments were performed on an Orbitrap Fusion Lumos instrument to detect SPLFGQYFVLENPGTIK (SEQ ID NO: 3675) (m / z = 955.5066) of the lightly labeled and heavily labeled peptides, and [ka] (At m / z=959.5137) was monitored for each sample. Next, the data was imported into Skyline 21.1 software (Pino LK et al. The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics. Mass Spectrom Rev. 2020 May;39(3):229-244.doi:10.1002 / mas.21540.Epub 2017 Jul 9), and the peak area of ​​the SPLFGQYFVLENPGTIK (SEQ ID NO: 3675) peptide from each sample was measured, along with the spiked SIL peptide. [ka] The peak area was normalized. GAPDH housekeeping protein was measured using the same starting tissue homogenate, precipitated with ice-cold acetone, then mixed at 1250 rpm for 10 minutes and centrifuged at 3220 × g for 15 minutes. The supernatant was aspirated, the protein pellet was washed with methanol, dissolved in 50 mM ammonium bicarbonate buffer containing 10 μg of trypsin, and digested overnight at 37°C with mixing at 1000 rpm. The digestion reaction was stopped with 20% formic acid and injected for LC-MS / MS analysis monitoring for the GAPDH peptide: LISWYDNEFGYSNR (SEQ ID NO: 3676) at 588.61 and 743.35 m / z. The peak area of ​​the GAPDH peptide was integrated using SCIEX Analyst software. Protein expression for each sample was normalized by taking the ratio of the concentration of the target gene (mARC1) determined to the SIL peptide over the concentration of the housekeeping gene (GAPDH). Next, the percentage of mARC1 protein expression after siRNA administration (days 14, 30, and 44) ​​was calculated for each animal replication per treatment group compared to pre-treatment (days -13 or -7), and this was expressed as the pre-treatment residual percentage. Finally, the percentage of mARC1 protein expression silencing was calculated by subtracting the pre-treatment residual percentage from 100%. Both the pre-treatment residual and silencing percentages for the protein are summarized in Table 29. Double-stranded nominal D-2081 showed the greatest reduction in mARC1 liver protein expression in cynomolgus monkeys at day 14 post-administration, with silencing of 89±0.71% after a single subcutaneous injection. At day 30 post-administration, double-stranded nominal D-2081 and D-2241 reduced protein expression to <20% pre-treatment residual, with silencing of 82±7.8% and 87±11%, respectively, which were maintained or increased by day 44 post-administration.

[0293] [Table 98]

[0294] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. The disclosed invention is not limited to the specific methodologies, protocols, and materials described herein, and these are to be understood to be subject to change. Furthermore, the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the scope of the appended claims.

[0295] Those skilled in the art will be able to recognize or verify, through mere routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents shall be encompassed within the following claims.

Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence complementary to the mRNA sequence of mARC1, and the region comprises at least 19 consecutive nucleotides from the antisense sequence of Sequence ID No. 2949. An RNAi construct in which the sense strand includes a sequence complementary to the sequence of the antisense strand, such that the sense strand and the antisense strand form a double-stranded region of 19 to 25 base pairs in length.

2. The RNAi construct according to claim 1, wherein the double-stranded region is 19 to 21 base pairs long.

3. The RNAi construct according to claim 1, wherein the sense strand and the antisense strand are each independently 19 to 30 nucleotides long.

4. The RNAi construct according to claim 3, wherein the sense strand and the antisense strand are each independently 19 to 23 nucleotides long.

5. The RNAi construct according to claim 1, comprising one or two blunt ends.

6. The RNAi construct according to claim 1, comprising one or two nucleotide overhangs of one to four unpaired nucleotides.

7. The RNAi construct according to claim 6, wherein the nucleotide overhang has two unpaired nucleotides.

8. The RNAi construct according to claim 6, comprising a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' ends of both the sense strand and the antisense strand.

9. The RNAi construct according to claim 1, comprising one or more modified nucleotides.

10. The RNAi construct according to claim 9, wherein the one or more modified nucleotides are a 2'-fluoromodified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a deoxyribonucleotide, or a combination thereof.

11. The RNAi construct according to claim 9, wherein all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.

12. The RNAi construct according to claim 11, wherein the modified nucleotide is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a combination thereof.

13. The RNAi construct according to claim 1, wherein the sense strand contains a debased nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' and 5' ends.

14. The RNAi construct according to claim 13, wherein the debasalized nucleotide is bound to an adjacent nucleotide via a 3'-3' nucleotide bond or a 5'-5' nucleotide bond.

15. The RNAi construct according to claim 9, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include one or more phosphorothioate nucleotide interlinks.

16. The RNAi construct according to claim 15, wherein the antisense strand includes two consecutive phosphorothioate nucleotide bonds between both the 3' and 5' terminal nucleotides.

17. The RNAi construct according to claim 15, wherein the sense strand includes a single phosphorothioate nucleotide bond between the terminal nucleotides at the 3' end.

18. The RNAi construct according to claim 15, wherein the sense strand includes two consecutive phosphorothioate nucleotide bonds between the terminal nucleotides at the 3' end.

19. The RNAi construct according to claim 1, wherein the antisense strand comprises or consists of a sequence selected from any one of the antisense sequences SEQ ID NOs: 2949, 1058, 1059, 1060, 2459, 2460, 2461, 2954, 2956, 2957, 3335, 3336, 3338, 3482, 3484, and 3485.

20. The RNAi construct according to claim 19, wherein the sense strand comprises or consists of a sequence selected from any one of the sense sequences SEQ ID NOs: 391, 3076, 389, 390, 1727, 1728, 1729, 2824, 3077, 3079, and 3192.

21. (i) The sense strand contains or consists of the sequence of sequence number 391, and the antisense strand contains or consists of the sequence of sequence number 2949, (ii) The sense strand contains or consists of the sequence of sequence number 391, and the antisense strand contains or consists of the sequence of sequence number 1060, (iii) The sense strand comprises or consists of the sequence of sequence number 389, and the antisense strand comprises or consists of the sequence of sequence number 1058, (iv) The sense strand contains or consists of the sequence of sequence number 390, and the antisense strand contains or consists of the sequence of sequence number 1059, (v) The sense strand contains or consists of the sequence of sequence number 389, and the antisense strand contains or consists of the sequence of sequence number 2954, (vi) The sense strand contains or consists of the sequence of sequence number 390, and the antisense strand contains or consists of the sequence of sequence number 2956, or (vii) The sense strand comprises or consists of the sequence of sequence number 2824, and the antisense strand comprises or consists of the sequence of sequence number 2957. The RNAi construct according to claim 1.

22. (i) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3076, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3472. (ii) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 1729, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 2461, (iii) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3076, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3335, (iv) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 1727, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 2459. (v) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3079, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3338. (vi) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 1728, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 2460. (vii) The sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3077, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 3336. (viiii) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3079, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3482, (ix) The sense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3077, and the antisense strand contains or consists of a sequence of modified nucleotides according to SEQ ID NO: 3484, or (x) The sense strand comprises or consists of a sequence of modified nucleotides according to Sequence ID No. 3192, and the antisense strand comprises or consists of a sequence of modified nucleotides according to Sequence ID No. 3485. The RNAi construct according to claim 21.

23. The RNAi construct according to claim 1, wherein the RNAi construct is D-2241, D-1389, D-2077, D-1387, D-2080, D-1388, D-2078, D-2256, D-2258, or D-2259.

24. The RNAi construct according to claim 23, wherein the RNAi construct is D-2241, D-1389, or D-2077.

25. The RNAi construct according to claim 24, which is D-2241.

26. The RNAi construct according to claim 1, further comprising a ligand.

27. The RNAi construct according to claim 26, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folic acid moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or an antigen-binding fragment thereof.

28. The RNAi construct according to claim 26, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

29. The RNAi construct according to claim 28, wherein the ligand comprises a polyvalent galactose moiety or a polyvalent N-acetyl-galactosamine moiety.

30. The RNAi construct according to claim 29, wherein the polyvalent galactose moiety or polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent.

31. The ligand is 【Chemistry 1】 The RNAi construct according to claim 26, comprising the structure.

32. The RNAi construct according to claim 26, wherein the ligand is optionally covalently bound to the sense strand via a linker.

33. The RNAi construct according to claim 32, wherein the ligand is covalently bound to the 5' end of the sense strand.

34. The RNAi construct further comprises a ligand covalently bound to the 5' end of the sense strand via a linker, wherein the ligand and linker are given formula VII: 【Chemistry 2】 (In the equation, X = O or S) The RNAi construct according to claim 22, having the structure.

35. The RNAi construct according to claim 34, wherein X = S.

36. An RNAi construct comprising a sense strand and an antisense strand, The sense strand consists of a sequence of modified nucleotides according to SEQ ID NO: 3076, and the antisense strand consists of a sequence of modified nucleotides according to SEQ ID NO: 3472. The RNAi construct further comprises a ligand covalently bound to the 5' end of the sense strand via a linker, wherein the ligand and linker are given formula VII: 【Transformation 3】 (In the equation, X = S) An RNAi construct having the structure.

37. A pharmaceutical composition comprising an RNAi construct according to any one of claims 1 to 36 and a pharmaceutically acceptable carrier or excipient.

38. A composition for reducing the expression of mARC1 protein in a patient in need, comprising the RNAi construct according to any one of claims 1 to 36.

39. The composition according to claim 38, wherein the patient has been diagnosed with or is at risk of having cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or cirrhosis.

40. A composition for reducing serum cholesterol in a patient in need, comprising the RNAi construct according to any one of claims 1 to 36.

41. The composition according to claim 40, wherein the serum cholesterol is non-HDL cholesterol or LDL cholesterol.

42. A composition comprising an RNAi construct according to any one of claims 1 to 36 for treating, preventing or reducing the risk of developing fatty liver disease in patients in need thereof.

43. The composition according to claim 42, wherein the fatty liver disease is non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

44. The composition according to claim 42, wherein the patient is diagnosed with type 2 diabetes, or is diagnosed with a metabolic disorder, or is obese.

45. The composition according to claim 42, wherein the patient has high levels of non-HDL cholesterol or triglycerides.

46. A composition comprising the RNAi construct described in any one of claims 1 to 36, for treating, preventing or reducing hepatic fibrosis in a patient in need thereof.

47. The composition according to claim 46, wherein the patient has been diagnosed with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

48. Use of an RNAi construct according to any one of claims 1 to 36 in the preparation of a pharmaceutical for reducing serum cholesterol in a patient in need thereof.

49. The use according to claim 48, wherein the serum cholesterol is non-HDL cholesterol or LDL cholesterol.

50. Use of an RNAi construct according to any one of claims 1 to 36 in the preparation of a pharmaceutical product for treating, preventing, or reducing the risk of developing fatty liver disease in patients in need thereof.

51. The use according to claim 50, wherein the fatty liver disease is non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

52. Use of an RNAi construct according to any one of claims 1 to 36 in the preparation of a pharmaceutical product for treating, preventing or reducing liver fibrosis in a patient in need thereof.

53. The use according to claim 52, wherein the patient has been diagnosed with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

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