DOUBLE-STRANDED NUCLEIC ACID INHIBITOR MOLECULES CONTAINING A TRILOOP
Provided herein are double-stranded nucleic acid inhibitor molecules having a sense strand with a stem loop structure and an antisense strand, where the loop portion of the stem loop structure is a triloop. Also provided are methods and compositions for reducing target gene expression and methods and compositions for treating a disease of interest.
1 . A double-stranded nucleic acid inhibitor molecule, comprising:
a sense strand comprising 20-65 nucleotides and having a first region (R1) and a second region (R2);
an antisense strand comprising 15-40 nucleotides, wherein the sense strand and antisense strand are separate strands; and
a first duplex (D1) formed by the first region of the sense strand and the antisense strand, wherein the first duplex has a length of 15-40 base pairs;
wherein the second region of the sense strand comprises a first subregion (S1), a second subregion (S2) and a triloop (triL) that joins the first and second regions, wherein the first and second regions form a second duplex (D2).
2 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the triloop has a nucleotide sequence of GAA.
3 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the sense strand has 22-65, 25-39, or 27-35 nucleotides.
4 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the antisense strand has 20-24 or 20-22 nucleotides.
5 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the nucleotide immediately adjacent to the 5′-end of the triloop is a C and the nucleotide immediately adjacent to the 3′-end of the triloop is a G.
6 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the antisense strand has a single stranded overhang of 1-4 nucleotides at its 3′-end.
7 . The double-stranded nucleic acid inhibitor molecule of claim 6 , wherein the single stranded overhang is 2 nucleotides in length.
8 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the first duplex has a length of 18-30, 18-24, or 20-22 base pairs.
9 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the second duplex has a length of 2-6 base pairs.
10 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the second duplex comprises at least one T m -increasing nucleotide, such as a bicyclic nucleotide.
11 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the second duplex has a length of 2 or 3 base pairs.
12 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the first region of the sense strand is 20 nucleotides in length and the second region of the sense strand is 7-15 nucleotides in length;
wherein the first duplex formed by the first region of the sense strand and the antisense strand has a length of 20 base pairs;
wherein the second duplex formed by the first and second nucleic acids of the second region of the sense strand has a length of 2-6 base pairs; and
wherein the antisense strand is 22 nucleotides in length and has a single-stranded overhang of two nucleotides at its 3′-end.
13 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein each nucleotide in the second duplex is a T m -increasing nucleotide.
14 . The double-stranded nucleic acid inhibitor molecule of claim 13 , wherein the second duplex has a length of 6 base pairs.
15 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the second duplex comprises at least one T m -increasing nucleotide is a bicyclic nucleotide chosen from one or more of the following:
wherein B is a nucleobase, R 2 is H or CH 3 and W a and W b are each independently, H, OH,
a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the bicyclic nucleotide to another nucleotide or to an oligonucleotide and wherein at least one of W a or W b is an internucleotide linking group attaching the bicyclic nucleotide to an oligonucleotide.
16 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the second duplex comprises at least one bicyclic nucleotide that comprises a first ring, wherein the first ring is a furanosyl, and a bridge that connects the 2′-carbon and the 4′-carbon of the furanosyl to form a second ring.
17 . The double-stranded nucleic acid inhibitor molecule of claim 16 , wherein the bridge that connects the 2′-carbon and the 4′-carbon of the furanosyl is selected from the group consisting of:
a) 4′-CH 2 —O—N(R)-2′ and 4′-CH 2 —N(R)—O-2′, wherein R is H, C 1 -C 12 alkyl, or a protecting group, including, for example, 4′-CH 2 —NH—O-2′ (also known as BNA NC ) or 4′-CH 2 —N(CH 3 )—O-2′ (also known as BNA NC [NMe]);
b) 4′-CH 2 -2; 4′—(CH 2 ) 2 -2; 4′—(CH 2 ) 3 -2; 4′—(CH 2 )—O-2′ (also known as LNA); 4′-(CH 2 )—S-2; 4′—(CH 2 ) 2 —O-2′ (also known as ENA); 4′-CH(CH 3 )—O-2′ (also known as cEt); and 4′-CH(CH 2 OCH 3 )—O-2′ (also known as cMOE), and analogs thereof;
c) 4′-C(CH 3 )(CH 3 )—O-2′ and analogs thereof;
d) 4′-CH 2 —N(OCH 3 )-2′ and analogs thereof;
e) 4′-CH 2 —O—N(CH 3 )-2′ and analogs thereof;
f) 4′-CH 2 —C(H)(CH 3 )-2′ and analogs thereof; and
g) 4′-CH 2 —C(═CH 2 )-2′ and analogs thereof.
18 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the triloop comprises at least one ligand conjugated nucleotide.
19 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the triloop comprises at least two ligand conjugated nucleotides.
20 . The double-stranded nucleic acid inhibitor molecule of claim 18 , wherein the ligand is a GalNAc.
21 . The double-stranded nucleic acid inhibitor molecule of claim 20 , wherein the GalNAc is conjugated to the nucleotide at the 2′-position of the sugar moiety.
22 . The double-stranded nucleic acid inhibitor molecule of claim 1 , further comprising a 5′-phosphate mimic at the 5′-terminus of the sense strand and/or the antisense strand.
23 . The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the double stranded the nucleic acid inhibitor molecule is formulated with a lipid nanoparticle.
24 . The double-stranded nucleic acid inhibitor molecule of claim 23 , wherein the lipid nanoparticle comprises core lipids and envelope lipids, wherein the core lipids comprise a first cationic lipid and a first pegylated lipid and wherein the envelope lipids comprise a second cationic lipid, a neutral lipid, a sterol, and a second pegylated lipid.
25 . The double-stranded nucleic acid inhibitor molecule of claim 24 , wherein the first cationic lipid is DL-048, the first pegylated lipid is DSG-MPEG, the second cationic lipid is DL-103, the neutral lipid is DSPC, the sterol is cholesterol, and the second pegylated lipid is DSPE-MPEG.
26 . A pharmaceutical composition comprising a therapeutically effective amount of the double-stranded nucleic acid inhibitor molecule of claim 1 and a pharmaceutically acceptable excipient.
27 . A method for reducing expression of a target gene in a subject comprising administering the double-stranded nucleic acid inhibitor molecule or pharmaceutical composition of claim 1 to a subject in need thereof in an amount sufficient to reduce expression of the target gene.
28 . The method of claim 27 , wherein the administering comprises intravenous, intramuscular, or subcutaneous administration.
29 . The method of claim 27 , wherein the subject is a human.