Double-stranded nucleic acid inhibitor molecules modified with Tm-increasing nucleotides
Provided herein are double-stranded nucleic acid inhibitor molecules having a sense strand with a stem loop structure and an antisense strand, where the stem portion of the stem loop structure contains one or more T m -increasing nucleotides. 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 21-66 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;
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 tetraloop (L) comprising at least one ligand conjugated nucleotide that joins the first and second subregions, wherein the first and second subregions form a second duplex (D2); and
wherein the second duplex has a length of 1-3 base pairs, wherein the second duplex contains 6 bicyclic nucleotides that form 3 base pairs, 4 bicyclic nucleotides that form 2 base pairs, 2 bicyclic nucleotides that form 1 base pair, 6 nucleotides that form 3 base pairs and include 3 unpaired bicyclic nucleotides, 6 nucleotides that form 3 base pairs and include 2 unpaired bicyclic nucleotides, or 6 nucleotides that form 3 base pairs and include 1 unpaired bicyclic nucleotide, and
wherein every nucleotide on the sense strand and antisense strand is modified at the 2′-carbon of the sugar moiety with a 2′-F or a 2′-OMe, except for the bicyclic nucleotides in the second duplex and the nucleotides in the tetraloop that are conjugated to a sugar ligand moiety.
2. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the sense strand has 24-35, 28-35, or 26-30 nucleotides.
3. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the antisense strand has 18-24, 21-23, or 22 nucleotides.
4. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the antisense strand has a single stranded overhang of 1-6 nucleotides at its 3′-end.
5. The double-stranded nucleic acid inhibitor molecule of claim 4 , wherein the single stranded overhang is 2 nucleotides in length.
6. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the first duplex has a length of 18-30, 18-24, or 19-21 base pairs.
7. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the sense strand is between 24-35 nucleotides in length, the antisense strand is between 20-24 nucleotides in length, the first duplex has a length of 18-24 base pairs.
8. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the sense strand is between 24-30 nucleotides in length, the antisense strand is between 20-24 nucleotides in length, the first duplex has a length of 18-24 base pairs.
9. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the second duplex has a length of 3 base pairs.
10. 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 10, 8, or 6 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 tetraloop is 4 nucleotides in length; and
wherein the antisense strand is 22 nucleotides in length and has a single-stranded overhang of two nucleotides at its 3′-end.
11. The double-stranded nucleic acid inhibitor molecule of claim 10 , wherein the second region of the sense strand is 6 nucleotides in length and the second duplex has a length of 1 base pair.
12. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein each nucleotide in the second duplex is the bicyclic nucleotide.
13. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the tetraloop is an RNA tetraloop selected from UNCG, GNRA, or CUUG, A/UGNN, GGUG, RNYA, or AGNN or a DNA tetraloop selected from d(GNAB), d(CNNG), or d(TNCG).
14. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the tetraloop has the sequence GAAA.
15. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide has the structure of Formula I, II, III, IV, Va, or Vb.
16. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide has the structure of Formula Ia, Ib, Ic, Id, Ie, or If.
17. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide has the structure of one or more of Formula IIa, IIb, IIc, or IId.
18. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide has the structure of one or more of Formula Ma or IIIb.
19. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide has the structure of one or more of Formula IVa or IVb.
20. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide is selected from the group consisting of:
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.
21. The double-stranded nucleic acid inhibitor molecule of claim 20 , wherein the bicyclic nucleotide is:
wherein B is a nucleobase, R 2 is 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.
22. The double-stranded nucleic acid inhibitor molecule of claim 20 , wherein the bicyclic nucleotide is:
wherein B is a nucleobase, R 2 is 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.
23. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the bicyclic nucleotide 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.
24. The double-stranded nucleic acid inhibitor molecule of claim 23 , 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.
25. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the tetraloop comprises two, three, or four ligand conjugated nucleotides.
26. The double-stranded nucleic acid inhibitor molecule of claim 1 , wherein the ligand is a GalNAc.
27. The double-stranded nucleic acid inhibitor molecule of claim 26 , wherein the GalNAc is conjugated to the nucleotide at the 2′-position of the sugar moiety.
28. 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.
29. 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.
30. The double-stranded nucleic acid inhibitor molecule of claim 29 , 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.
31. The double-stranded nucleic acid inhibitor molecule of claim 30 , 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.
32. A pharmaceutical composition comprising a therapeutically effective amount of the double-stranded nucleic acid inhibitor molecule of claim 1 and a pharmaceutically acceptable excipient.
33. 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 32 to a subject in need thereof in an amount sufficient to reduce expression of the target gene.
34. The method of claim 33 , wherein the administering comprises intravenous, intramuscular, or subcutaneous administration.
35. The method of claim 33 , wherein the subject is a human.
36. The double-stranded nucleic acid inhibitor molecule of claim 24 , wherein each nucleotide in the second duplex is the bicyclic nucleotide.