Formats for organic compounds for use in RNA interference
The disclosure relates to compositions comprising a RNAi agent having a novel format including a spacer subunit. The disclosure relates to compositions comprising a RNAi agent having a novel format: an 18-mer format with at least one internal spacer. These RNAi agents comprise a first and a second 18-mer strand, wherein the first strand is 18 ribonucleotides or 18 total ribonucleotides and spacer subunit(s), and the second strand is 18 total ribonucleotides and spacer subunit(s), wherein: each spacer subunit consists of: (a) a phosphate or modified internucleoside linker and (b) a spacer; a spacer subunit can be at any position in the strand; the two strands form a duplex with at least one blunt end; and the 3 end of one or both strands terminates in a phosphate or modified internucleoside linker and further comprises, in 5 to 3 order: a second spacer; a second phosphate or modified internucleoside linker, and a 3 end cap. In various embodiments, the RNAi agents comprise a first and a second strand, wherein each strand is a 30-mer or shorter, the first strand comprises ribonucleotides, and the second strand comprises ribonucleotides and one or more spacer subunit(s), wherein: each spacer subunit consists of: (a) a phosphate or modified internucleoside linker and (b) a spacer; a spacer subunit can be at any position in the strand. In some embodiments, the 3 end of both strands further comprise, in 5 to 3 order: a second spacer; a second phosphate or modified internucleoside linker; and a 3 end cap. The two strands can have the same or different spacers, phosphates or modified internucleoside linkers, and/or 3 end caps. The strands can be ribonucleotides, or, optionally, one or more nucleotide can be modified or substituted. Optionally, at least one nucleotide comprises a modified internucleoside linker. Optionally, the first two base-pairing nucleotides on the 3 end of the one or both strand are 2-MOE. Optionally, the RNAi agent can be modified on one or both 5 end. Optionally, the first or second strand is the sense strand, and the sense strand can comprise a 5 end cap which reduces the amount of the RNA interference mediated by this strand. Optionally, the RNAi agent is attached to a ligand. This format can be used to devise RNAi agents to a variety of different targets and sequences. The disclosure also relates to processes for making such compositions, and methods and uses of such compositions, e.g., to mediate RNA interference.
1. An RNAi agent comprising a first and a second 18-mer strand, wherein
the first 18-mer strand is 18 total of:
(a) 13-17 ribonucleotides and/or modified ribonucleotides,
(b) at least one internal spacer subunit comprising ribitol, and optionally
(c) 0-4 of any of DNA, PNA, LNA, TNA, GNA, ANA, FANA, CeNa, HNA or UNA; and
the second 18-mer strand is 18 total of:
(a) 13-18 ribonucleotides and/or modified ribonucleotides, optionally
(b) at least one spacer subunit, and optionally
(c) 0-4 of any of DNA, PNA, LNA, TNA, GNA, ANA, FANA, CeNa, HNA or UNA;
wherein each spacer subunit is
(a) a phosphate or modified internucleoside linker and
(b) a spacer,
wherein the first and second 18-mer strands together form at least one blunt-end, and
wherein the 3′ end of at least one of the first or second 18-mer strands terminates in a phosphate or modified internucleoside linker which is optionally further substituted by,
(a) a first 3′ end cap; or
(b) in 5′ to 3′ order: a second spacer; a second phosphate or modified internucleoside linker; and a second 3′ end cap.
2. The RNAi agent of claim 1 wherein the 3′ end of at least one of the first and second 18-mer strands terminates in the phosphate or modified internucleoside linker which is further substituted by, in 5′ to 3′ order: the second spacer; the second phosphate or modified internucleoside linker; and the second 3′ end cap.
3. The RNAi agent of claim 1 wherein: the first 18-mer strand is 17 ribonucleotides and/or modified ribonucleotides and 1 internal spacer subunit; and the second 18-mer strand is 18 ribonucleotides and/or modified ribonucleotides.
4. The RNAi agent of claim 1 wherein the at least one spacer subunit of the second 18-mer strand is at any of positions 2 to 17, and the first and second 18-mer strands form a blunt-ended duplex.
5. The RNAi agent of claim 1 wherein at least one nucleotide comprises a modified internucleoside linker.
6. The RNAi agent of claim 1 wherein the RNAi agent is attached to a ligand.
7. The RNAi agent of claim 1 wherein the first 18-mer strand is 17 ribonucleotides and/or modified ribonucleotides and 1 internal spacer subunit; and the second 18-mer strand is 18 ribonucleotides and/or modified ribonucleotides, and wherein the internal spacer subunit comprises a ribitol.
8. The RNAi agent of claim 1 wherein the modified internucleoside linker of the spacer subunit of the second 18-mer strand, and/or at the 3′ end of the at least one of the first or second 18-mer strands, and/or the second modified internucleoside linker is selected from phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, and a compound of formula (I):
where R 3 is selected from O − , S − , NH 2 , BH 3 , CH 3 , C 1-6 alkyl, C 6-10 aryl, O-alkyl and O-aryl; and R 4 is selected from O, S, NH and CH 2 ; wherein said C 1-6 alkyl or C 6-10 aryl of R 3 are unsubstituted or substituted by one to three substituents each independently selected from halo, hydroxyl and NH 2 .
9. The RNAi agent of claim 1 , wherein the first and/or second 3′ end cap is: A compound formula Ia:
in which:
X is the attachment to the phosphate or modified internucleoside linker at the 3′ end of the at least one of the first or second 18-mer strands, or
the attachment to the second phosphate or modified internucleoside linker of the at least one of the first or second 18-mer strand;
Y is selected from CH and N;
m is selected from 0 and 1;
p is selected from 1, 2 and 3;
R 3 is selected from hydrogen, 2-(hydroxy-methyl)-benzyl, 3-(hydroxy-methyl)-benzyl and succinate, or is attached to a solid support; wherein the (CH 2 ) m —O—R 3 moiety is attached to the phenyl ring at position 3 or 4;
R 4 is hydrogen;
R 5 is hydrogen; or
R 4 and R 5 , together with the phenyl rings to which R 4 and R 5 are attached, form 6H-benzo[c]chromene;
or
A formula Ib:
in which:
X is the attachment to the phosphate or modified internucleoside linker at the 3′ end of the at least one of the first or second 18-mer strands, or
the attachment to the second phosphate or modified internucleoside linker of the at least one of the first or second 18-mer strands;
q is selected from 0, 1 and 2;
R 6 is selected from phenyl which is unsubstituted or substituted with benzoxy;
R 7 is selected from hydrogen and hydroxy-ethyl, wherein if R 7 is hydroxy-ethyl, the hydroxyl can be optionally functionalized as succinate or attached to a solid support;
R 8 is selected from hydrogen and methoxy;
Y 1 is selected from CH and N; and
Y 2 is selected from N and CR 9 ; wherein R 9 is selected from hydrogen and methyl.
10. The RNAi agent of claim 9 , wherein the first and/or second 3′ end cap is selected from any of:
In which:
X is the attachment to the phosphate or modified internucleoside linker at the 3′ end of the at least one of the first or second 18-mer strands, or
the attachment to the second phosphate or modified internucleoside linker of the at least one of the first or second 18-mer strands
q is selected from 1 and 2; or
wherein the first and/or second 3′ end cap is selected from any of: triethylene glycol, cyclohexyl, phenyl, lithochol (lithocholic acid), or adamantane.
11. The RNAi agent of claim 1 wherein the first and second 3′ end caps on the first and second strands are different.
12. The RNAi agent of claim 1 wherein one or more nucleotide are modified or substituted.
13. The RNAi agent of claim 1 wherein the RNAi agent comprises at least one non-natural nucleobase.
14. The RNAi agent of claim 13 wherein the non-natural nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl.
15. The RNAi agent of claim 1 wherein the first two base-pairing nucleotides on the 3′ end of the first and/or second strand are modified.
16. The RNAi agent of claim 1 wherein the first two base-pairing nucleotides on the 3′ end of the first and/or second strand are 2′-MOE.
17. The RNAi agent of claim 1 wherein the 3′ end of the first and/or second strands terminates in a modified internucleoside linker.
18. The RNAi agent of claim 1 wherein at least one 18-mer strand comprises 1-3 2′-deoxynucleotides.
19. The RNAi agent of claim 1 wherein the RNAi agent is modified on one or both 5′ ends.
20. A pharmaceutical composition comprising the RNAi agent of claim 1 and a pharmaceutically acceptable carrier.
21. A method for reducing the level and/or activity of a target gene in a cell, comprising the step of introducing into the cell the RNAi agent of claim 1 , wherein the RNAi agent targets the target gene.