Modified rna agents with reduced off-target effect

Patent metadata

Field	Value
Publication number	US-2025207131-A1
Application number	US-202418968749-A
Country	US
Kind code	A1
Filing date	Dec 4, 2024
Priority date	May 16, 2018
Publication date	Jun 26, 2025
Grant date	—

Abstract

One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The antisense strand of the dsRNA molecule comprises at least one thermally destabilizing nucleotide occurring at a seed region; the dsRNA comprises at least four 2′-fluoro modifications, and the sense strand of the dsRNA molecule comprises ligand, wherein the ligand is an ASGPR ligand. Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA molecules suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA molecules, e.g., for the treatment of various disease conditions.

First claim

We claim:

1 . A double-stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, each strand having 18 to 35 nucleotides, wherein
the dsRNA molecule has a duplex region of 17-30 nucleotide pairs in length; the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference; the antisense strand comprises a thermally destabilizing modification at position 6 or 7 counting from the 5′-end of the antisense strand; and the thermally destabilizing modification is of the formula,

 and
wherein the dsRNA molecule is optionally conjugated to a ligand.

2 . The dsRNA molecule according to claim 1 , wherein the duplex region is 19-23 base pairs in length.

3 . The dsRNA molecule according to claim 1 , comprising at least one ligand conjugated to the sense strand.

4 . The dsRNA molecule according to claim 3 , wherein at least one ligand is a targeting ligand.

5 . The dsRNA molecule according to claim 4 , wherein the targeting ligand is an ASGPR ligand that is conjugated to the 3′-end or 5′-end of the sense strand.

6 . The dsRNA molecule according to claim 5 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

7 . The dsRNA molecule of claim 6 , wherein the ASGPR ligand is:

8 . The dsRNA molecule according to claim 4 , wherein the targeting ligand is a lipid, a cell targeting peptide, or a ligand that improves nuclease resistance of the dsRNA molecule.

9 . The dsRNA molecule of claim 1 , having a melting temperature of about 55° C. to about 67° C.

10 . The dsRNA molecule of claim 1 , having a blunt end at 5′-end of the antisense strand.

11 . The dsRNA molecule of claim 10 , having a blunt end at 3′-end of the antisense strand.

12 . The dsRNA molecule of claim 10 , having a single-stranded overhang of 1-10 nucleotides in length at the 3′-end of the antisense strand.

13 . The dsRNA molecule of claim 1 , wherein each strand is 19-25 nucleotides in length.

14 . The dsRNA molecule of claim 1 , wherein the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.

15 . The dsRNA molecule of claim 1 , wherein the antisense strand comprises only 2, 3, 4, 5, or 6 2′-fluoro modifications.

16 . The dsRNA molecule according to claim 15 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand, counting from the 5′ end of the antisense strand.

17 . The dsRNA molecule of claim 15 , wherein the antisense strand has 2′-F modifications at positions 2, 14, and 16; or positions 2, 6, 14, and 16; or positions 2, 6, 8, 9, 14, and 16, counting from the 5′ end of the antisense strand.

18 . The dsRNA molecule of claim 1 , wherein the sense strand comprises only 2, 3, 4 or 5 2′-fluoro modifications.

19 . The dsRNA molecule of claim 18 , wherein the sense strand has 2′-F modifications at positions complimentary to positions 11, 12, and 15; or positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand.

20 . The dsRNA molecule of claim 1 , wherein the antisense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages, and wherein at least the terminal two nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate or methylphosphonate internucleotide linkages.

21 . The dsRNA molecule of claim 20 , wherein the terminal three nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate internucleotide linkages.

22 . The dsRNA molecule of claim 1 , wherein the sense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages, and wherein at least the terminal two nucleotides at one end or both ends of the sense strand are linked through phosphorothioate or methylphosphonate internucleotide linkages.

23 . The dsRNA molecule of claim 1 , wherein the terminal three nucleotides at one end or both ends of the sense strand are linked through phosphorothioate internucleotide linkages.

24 . The dsRNA molecule of claim 1 , the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

25 . The dsRNA molecule of claim 1 , wherein the antisense strand comprises a 5′-(E)-vinyl phosphonate (VP).

26 . The dsRNA molecule of claim 1 , wherein the thermally destabilizing modification is at position 6 of the antisense strand, counting from the 5′end of the antisense strand.

27 . The dsRNA molecule of claim 1 , wherein the thermally destabilizing modification is at position 7 of the antisense strand, counting from the 5′end of the antisense strand.

28 . A pharmaceutical composition comprising the dsRNA agent according to claim 1 , alone or in combination with a pharmaceutically acceptable carrier or excipient.

29 . A method for silencing a target gene in a cell, the method comprising a step of introducing the dsRNA molecule of claim 1 into the cell.

30 . A method for delivering polynucleotide to a specific target in a subject by administering the dsRNA agent according to claim 1 .

31 . The method of claim 28 , wherein said administering step is carried out by an administration means comprising intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or combinations thereof.

32 . A compound of the formula,

wherein Ph is phenyl and DMTr is 4,4′-dimethoxytrityl.

Assignees

• Alnylam Pharmaceuticals Inc

Inventors

• Matsuda Shigeo
• Schlegel Mark K
• Janas Maja
• Jadhav Vasant R
• Maier Martin
• Charisse Klaus
• Manoharan Muthiah
• Rajeev Kallathottathil G
• Nair Jayaprakash K

Classifications

• C12N2310/53

  partially self-complementary or closed · CPC title

• C12N2310/344

  Position-specific modifications, e.g. on every purine, at the 3'-end · CPC title

• C12N2310/33

  of the base · CPC title

• C12N2310/323

  modified ring structure · CPC title

• C12N2310/318

  where the PO2 is completely replaced, e.g. MMI or formacetal · CPC title