Carbohydrate conjugates as delivery agents for oligonucleotides
The present invention provides a modified RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand targets an HBV mRNA; all of the nucleotide sugars of the RNAi agent are 2′-modified; and at least one of the sense and antisense strands is conjugated to at least one targeting ligand that comprises two or more building blocks connected to a biantennary or triantennary branched linker, optionally via one or more linking groups.
1. A modified RNAi agent comprising a sense strand and an antisense strand, wherein:
the antisense strand targets an HBV mRNA;
all of the nucleotide sugars of the RNAi agent are 2′-modified, wherein each 2′-modification is independently selected from the group consisting of 2′-H, 2′-(2-methoxyethoxy), 2′-OCH 3 , 2′-O-allyl, 2′—C— allyl, and 2′-fluoro; and
at least one of the sense and antisense strands is conjugated to at least one targeting ligand that consists of two or more building blocks connected to a biantennary or triantennary branched linker,
wherein the two or more building blocks are selected from the group consisting of:
wherein the biantennary branched linker consists of a branched carbon atom connected to the building blocks through one or more of —C(O)—, —NH—, —O—, —NHC(O)—, and —(CH 2 ) x — where x is 1-4, and
wherein the triantennary branched linker consists of a branched carbon atom connected to the building blocks through one or more of —C(O)—, —NH—, —O—, —NHC(O)—, and —(CH 2 ) x — where x is 1-4.
2. The modified RNAi agent of claim 1 , wherein the targeting ligand is at the 3′-end of the sense strand.
3. The modified RNAi agent of claim 1 , wherein the targeting ligand is at the 5′-end of the sense strand.
4. The modified RNAi agent of claim 1 , wherein each 2′-modification is independently selected from the group consisting of 2′-OCH 3 and 2′-fluoro.
5. The modified RNAi agent of claim 1 , wherein the targeting ligand consists of three building blocks connected to a biantennary or triantennary branched linker.
6. The modified RNAi agent of claim 5 , wherein the targeting ligand is prepared by reacting a building block precursor compound selected from the group consisting of:
or a salt thereof, wherein each hydroxyl group of the building block precursor compound is protected with a hydroxyl protecting group, with a biantennary or triantennary branched linker compound that contains a terminal carboxylic acid group in each branch of the branched linker compound, via an amidation reaction.
7. The modified RNAi agent of claim 6 , wherein the building block precursor compound is selected from the group consisting of:
8. The modified RNAi agent of claim 5 , wherein the targeting ligand is prepared by reacting a building block precursor compound having the formula,
or a salt thereof, wherein each hydroxyl group of the building block precursor compound is protected with a hydroxyl protecting group, with a biantennary or triantennary branched linker compound that contains a terminal carboxylic acid group in each branch of the branched linker compound, via an amidation reaction.
9. The modified RNAi agent of claim 8 , wherein the building block precursor compound is
10. The modified RNAi agent of claim 5 , wherein the targeting ligand is prepared by reacting a building block precursor compound having the formula,
or a salt thereof, wherein each hydroxyl group of the N-acetyl galactosamine moiety of the building block precursor compound is protected with a hydroxyl protecting group, with a biantennary or triantennary branched linker compound that contains a terminal hydroxyl group in each branch of the branched linker compound, via a dehydration reaction.
11. The modified RNAi agent of claim 10 , wherein the building block precursor compound is
12. The modified RNAi agent of claim 5 , wherein the targeting ligand is prepared by reacting a building block precursor compound selected from the group consisting of:
or a salt thereof, wherein each hydroxyl group of the building block precursor compound is protected with a hydroxyl protecting group, with a biantennary or triantennary branched linker compound that contains a terminal amine group in each branch of the branched linker compound, via an amidation reaction.
13. The modified RNAi agent of claim 12 , wherein the building block precursor compound is selected from the group consisting of:
14. The modified RNAi agent of claim 1 , wherein
the antisense strand has 3 or fewer mismatches with respect to the HBV mRNA; and
the sense strand that is sufficiently complementary with the antisense strand to form the double-stranded modified RNAi agent.
15. The modified RNAi agent of claim 14 , wherein each of the sense and antisense strands is independently 17 to 25 nucleotides in length.
16. The modified RNAi agent of claim 15 , wherein the sense and antisense strands form a duplex region of 17 to 23 nucleotide pairs in length.
17. The modified RNAi agent of claim 15 , wherein the sense and antisense strands form a duplex region of 21 nucleotide pairs in length.
18. The modified RNAi agent of claim 16 , comprising no overhang, or one or two 3′ overhangs of 2 or 3 nucleotides in length.
19. The modified RNAi agent of claim 1 , wherein the sense or antisense strand, or both, comprise a terminal modification suitable for modulating resistance to degradation.
20. The modified RNAi agent of claim 1 , comprising one or more phosphorothioate modifications within one or both of the 3′ or 5′ terminal regions of the sense strand, the antisense strand, or both strands.
21. The modified RNAi agent of claim 1 , wherein the sense strand is conjugated with a targeting ligand that consists of two or three building blocks connected to a biantennary or triantennary branched linker, wherein the building blocks are selected from the group consisting of:
22. A pharmaceutical composition comprising the modified RNAi agent of claim 1 in combination with a pharmaceutically acceptable carrier or excipient.