Compositions and methods for inhibiting mitochondria amidoxime reducing component 1 (marc1) expression

Abstract

Oligonucleotides are provided herein that inhibit MARC1 expression. Also provided are compositions including the same and uses thereof, particularly uses relating to treating diseases, disorders and/or conditions associated with MARC1 expression.

1 . An RNAi oligonucleotide for reducing MARC1 expression, comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a region of complementarity to a MARC1 mRNA target sequence of any one of SEQ ID NOs: 1-384, and wherein the region of complementarity is at least 15 contiguous nucleotides in length differing by no more than 3 nucleotides from the MARC1 mRNA target sequence. 
     
     
         2 . The RNAi oligonucleotide of  claim 1 , comprising:
 (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a MARC1 mRNA target sequence, wherein the region of complementarity is selected from SEQ ID NOs: 385-768, and   (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3′ terminus of the antisense strand.   
     
     
         3 . The RNAi oligonucleotide of  claim 1 , wherein:
 (i) the sense strand is 15 to 50 or 18 to 36 nucleotides in length,   (ii) the antisense strand is 15 to 30 nucleotides in length, and   (iii) the duplex region is at least 19 nucleotides or at least 20 nucleotides in length.   
     
     
         4 . The RNAi oligonucleotide of  claim 3 , wherein the sense strand is 36 nucleotides in length, and the antisense strand is 22 nucleotides in length. 
     
     
         5 . The RNAi oligonucleotide of  claim 1 , wherein the 3′ end of the sense strand comprises a stem-loop set forth as S1-L-S2, wherein
 (i) S1 is complementary to S2, and S1 and S2 are each 1-10 nucleotides in length and have the same length; and 
 (ii) L forms a loop between S1 and S2 of 3-5 nucleotides in length. 
 
     
     
         6 . The RNAi oligonucleotide of  claim 5 , wherein S1 and S2 are 6 nucleotides in length and L is a triloop or a tetraloop. 
     
     
         7 . The RNAi oligonucleotide of  claim 6 , wherein L is a tetraloop comprising the sequence of 5′-GAAA-3′. 
     
     
         8 . The RNAi oligonucleotide of  claim 5 , wherein the stem-loop comprises the sequence 5′-GCAGCCGAAAGGCUGC-3′ (SEQ ID NO: 1681). 
     
     
         9 . The RNAi oligonucleotide of  claim 5 , wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein:
 (a) each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide or lipid;   (b) the stem loop comprises one or more targeting ligands conjugated to one or more nucleotides of the stem loop;   (c) the one or more targeting ligands is conjugated to one or more nucleotides of the loop, optionally wherein the loop comprises 4 nucleotides numbered 1-4 from 5′ to 3′, wherein nucleotides at positions 2, 3, and 4 each comprise one or more targeting ligands, wherein the targeting ligands are the same or different;   (d) the targeting ligand is a hepatocyte targeting ligand and each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety, wherein the GalNAc moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; and/or   (e) the targeting ligand is a hepatocyte targeting ligand and up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety.   
     
     
         10 . The RNAi oligonucleotide of  claim 8 , wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, wherein:
 (a) each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide or lipid;   (b) the stem loop comprises one or more targeting ligands conjugated to one or more nucleotides of the stem loop;   (c) the one or more targeting ligands is conjugated to one or more nucleotides of the loop, optionally wherein the loop comprises 4 nucleotides numbered 1-4 from 5′ to 3′, wherein nucleotides at positions 2, 3, and 4 each comprise one or more targeting ligands, wherein the targeting ligands are the same or different;   (d) the targeting ligand is a hepatocyte targeting ligand and each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety, wherein the GalNAc moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; and/or   (e) the targeting ligand is a hepatocyte targeting ligand and up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety.   
     
     
         11 . The RNAi oligonucleotide of  claim 1 , wherein:
 (i) the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification, wherein:
 (a) the 2′-modification is a modification selected from 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid, optionally wherein the modification is selected from 2′-fluoro and 2′-O-methyl, optionally wherein all nucleotides of the oligonucleotide are modified, wherein the modification is 2′-fluoro and 2′-O-methyl; 
 (b) about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2′-fluoro modification; 
 (c) about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2′-fluoro modification; 
 (d) about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2′-fluoro modification; 
 (e) the sense strand comprises 36 nucleotides with positions 1-36 from 5′ to 3′, wherein positions 8-11 comprise a 2′-fluoro modification; 
 (f) the antisense strand comprises 22 nucleotides with positions 1-22 from 5′ to 3′, and wherein positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2′-fluoro modification; and/or 
 (g) the remaining nucleotides comprise a 2′-O-methyl modification, and/or 
   (ii) the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage, wherein:
 (a) the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5′ to 3′; and/or 
 (b) the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5′ to 3′, and/or 
   (iii) the antisense strand comprises a phosphorylated nucleotide at the 5′ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine, and/or   (iv) the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog selected from the group consisting of oxymethylphosphonate, vinylphosphonate and malonylphosphonate, and/or   (v) the antisense strand comprises an overhang sequence of one or more nucleotides in length at the 3′ terminus, and wherein the overhang comprises purine nucleotides, and optionally the overhang is selected from AA, GG, AG, and GA.   
     
     
         12 . The RNAi oligonucleotide of  claim 11 , wherein the phosphorylated nucleotide is uridine. 
     
     
         13 . The RNAi oligonucleotide of  claim 11 , wherein the phosphate analog is a 4′-phosphate analog comprising 5′-methoxyphosphonate-4′-oxy. 
     
     
         14 . The RNAi oligonucleotide of  claim 11 , wherein the overhang is GG. 
     
     
         15 . The RNAi oligonucleotide of  claim 9 , wherein:
 (i) the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification, wherein:
 (a) the 2′-modification is a modification selected from 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid, optionally wherein the modification is selected from 2′-fluoro and 2′-O-methyl, optionally wherein all nucleotides of the oligonucleotide are modified, wherein the modification is 2′-fluoro and 2′-O-methyl; 
 (b) about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2′-fluoro modification; 
 (c) about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2′-fluoro modification; 
 (d) about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2′-fluoro modification; 
 (e) the sense strand comprises 36 nucleotides with positions 1-36 from 5′ to 3′, wherein positions 8-11 comprise a 2′-fluoro modification; 
 (f) the antisense strand comprises 22 nucleotides with positions 1-22 from 5′ to 3′, and wherein positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2′-fluoro modification; and/or 
 (g) the remaining nucleotides comprise a 2′-O-methyl modification, and/or 
   (ii) the oligonucleotide comprises at least one modified internucleotide linkage, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage, wherein:
 (a) the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5′ to 3′; and/or 
 (b) the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5′ to 3′, and/or 
   (iii) the antisense strand comprises a phosphorylated nucleotide at the 5′ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine, and/or   (iv) the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, selected from the group consisting of oxymethylphosphonate, vinylphosphonate and malonylphosphonate, and/or   (v) the antisense strand comprises an overhang sequence of one or more nucleotides in length at the 3′ terminus and wherein the overhang comprises purine nucleotides and optionally the overhang is selected from AA, GG, AG and GA.   
     
     
         16 . The RNAi oligonucleotide of  claim 15 , wherein the phosphorylated nucleotide is uridine. 
     
     
         17 . The RNAi oligonucleotide of  claim 15 , wherein the phosphate analog is a 4′-phosphate analog comprising 5′-methoxyphosphonate-4′-oxy. 
     
     
         18 . The RNAi oligonucleotide of  claim 15 , wherein the overhang is GG. 
     
     
         19 . The RNAi oligonucleotide of  claim 1 , wherein the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of:
 (a) SEQ ID NOs: 1537 and 1573, respectively;   (b) SEQ ID NOs: 1538 and 1574, respectively;   (c) SEQ ID NOs: 1539 and 1575, respectively;   (d) SEQ ID NOs: 1540 and 1576, respectively;   (e) SEQ ID NOs: 1541 and 1577, respectively;   (f) SEQ ID NOs: 1542 and 1578, respectively;   (g) SEQ ID NOs: 1543 and 1579, respectively;   (h) SEQ ID NOs: 1544 and 1580, respectively;   (i) SEQ ID NOs: 1545 and 1581, respectively;   (j) SEQ ID NOs: 1546 and 1582, respectively;   (k) SEQ ID NOs: 1547 and 1583, respectively;   (l) SEQ ID NOs: 1548 and 1584, respectively;   (m) SEQ ID NOs: 1549 and 1585, respectively;   (n) SEQ ID NOs: 1550 and 1586, respectively;   (o) SEQ ID NOs: 1551 and 1587, respectively;   (p) SEQ ID NOs: 1552 and 1588, respectively;   (q) SEQ ID NOs: 1553 and 1589, respectively;   (r) SEQ ID NOs: 1554 and 1590, respectively;   (s) SEQ ID NOs: 1555 and 1591, respectively;   (t) SEQ ID NOs: 1556 and 1592, respectively;   (u) SEQ ID NOs: 1557 and 1593, respectively;   (v) SEQ ID NOs: 1558 and 1594, respectively;   (w) SEQ ID NOs: 1559 and 1595, respectively;   (x) SEQ ID NOs: 1560 and 1596, respectively;   (y) SEQ ID NOs: 1561 and 1597, respectively;   (z) SEQ ID NOs: 1562 and 1598, respectively;   (aa) SEQ ID NOs: 1563 and 1599, respectively;   (bb) SEQ ID NOs: 1564 and 1600, respectively;   (cc) SEQ ID NOs: 1565 and 1601, respectively;   (dd) SEQ ID NOs: 1566 and 1602, respectively;   (ee) SEQ ID NOs: 1567 and 1603, respectively;   (ff) SEQ ID NOs: 1568 and 1604, respectively;   (gg) SEQ ID NOs: 1569 and 1605, respectively; and   (hh) SEQ ID NOs: 1570 and 1606, respectively.   
     
     
         20 . The RNAi oligonucleotide of  claim 1 , wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of:
 (a) SEQ ID NOs: 1609 and 1645, respectively;   (b) SEQ ID NOs: 1610 and 1646, respectively;   (c) SEQ ID NOs: 1611 and 1647, respectively;   (d) SEQ ID NOs: 1612 and 1648, respectively;   (e) SEQ ID NOs: 1613 and 1649, respectively;   (f) SEQ ID NOs: 1614 and 1650, respectively;   (g) SEQ ID NOs: 1615 and 1651, respectively;   (h) SEQ ID NOs: 1616 and 1652, respectively;   (i) SEQ ID NOs: 1617 and 1653, respectively;   (j) SEQ ID NOs: 1618 and 1654, respectively;   (k) SEQ ID NOs: 1619 and 1655, respectively;   (l) SEQ ID NOs: 1620 and 1656, respectively;   (m) SEQ ID NOs: 1621 and 1657, respectively;   (n) SEQ ID NOs: 1622 and 1658, respectively;   (o) SEQ ID NOs: 1623 and 1659, respectively;   (p) SEQ ID NOs: 1624 and 1660, respectively;   (q) SEQ ID NOs: 1625 and 1661, respectively;   (r) SEQ ID NOs: 1626 and 1662, respectively;   (s) SEQ ID NOs: 1627 and 1663, respectively;   (t) SEQ ID NOs: 1628 and 1664, respectively;   (u) SEQ ID NOs: 1628 and 1665, respectively;   (v) SEQ ID NOs: 1630 and 1666, respectively;   (w) SEQ ID NOs: 1631 and 1667, respectively;   (x) SEQ ID NOs: 1632 and 1668, respectively;   (y) SEQ ID NOs: 1633 and 1669, respectively;   (z) SEQ ID NOs: 1634 and 1670, respectively;   (aa) SEQ ID NOs: 1635 and 1671, respectively;   (bb) SEQ ID NOs: 1636 and 1672, respectively;   (cc) SEQ ID NOs: 1637 and 1673, respectively;   (dd) SEQ ID NOs: 1638 and 1674, respectively;   (ee) SEQ ID NOs: 1639 and 1675, respectively;   (ff) SEQ ID NOs: 1640 and 1676, respectively;   (gg) SEQ ID NOs: 1641 and 1677, respectively; and   (hh) SEQ ID NOs: 1642 and 1678, respectively.   
     
     
         21 . A pharmaceutical composition comprising the RNAi oligonucleotide of  claim 20 , and a pharmaceutically acceptable carrier, delivery agent or excipient. 
     
     
         22 . A method of treating a disease or condition associated with MARC1 expression in hepatocytes selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and alcoholic steatohepatitis (ASH), comprising administering a therapeutically effective amount of the double stranded RNAi oligonucleotide of  claim 20 . 
     
     
         23 . A kit comprising the double stranded RNAi oligonucleotide of  claim 20 , a pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with MARC1 expression selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and alcoholic steatohepatitis (ASH).