US2023256001A1PendingUtilityA1

Modified rna agents with reduced off-target effect

Assignee: ALNYLAM PHARMACEUTICALS INCPriority: Nov 23, 2016Filed: Sep 30, 2022Published: Aug 17, 2023

Est. expiryNov 23, 2036(~10.4 yrs left)· nominal 20-yr term from priority

Inventors:Mark K. Schlegel Maja Janas Vasant Jadhav Muthiah Manoharan Kallanthottathil G. Rajeev Muthusamy Jayaraman Alexander V. Kel'In Shigeo Matsuda Klaus Charisse Jayaprakash K. Nair Martin Maier Alfica Sehgal Christopher Brown Christopher Theile Stuart Milstein

C12N 2310/351A61P 43/00C12N 2310/3533C12N 2310/14C12N 2310/322C12N 2320/53C12N 2310/31C12N 15/113A61K 31/713

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References

Claims

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.

Claims

exact text as granted — not AI-modified

What is claimed is: 
     
         1 . A double-stranded RNA (dsRNA) molecule capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein
 the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference;   the dsRNA comprises a duplex region of 12-40 base pairs in length;   the antisense strand comprises at least one thermally destabilizing modification of the duplex at one of positions 5, 6, 7 or 8, counting from the 5′-end of the antisense strand; and   the antisense strand comprises 2, 3, 4, 5 or 6 2′-fluoro modifications.   
     
     
         2 . The dsRNA molecule according to  claim 1 , wherein the thermally destabilizing modification of the duplex is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       wherein B is nucleobase, and *represents either R, S or racemic. 
     
     
         3 . The dsRNA molecule according to  claim 1 , wherein the thermally destabilizing modification of the duplex is 
       
         
           
           
               
               
           
         
       
       (GNA), and *represents either R, S or racemic. 
     
     
         4 . The dsRNA molecule according to  claim 3 , wherein the thermally destabilizing modification of the duplex is an (S)-GNA modification. 
     
     
         5 . The dsRNA molecule according to  claim 1 , wherein the thermally destabilizing modification of the duplex is an abasic modification, a mismatch with the opposing nucleotide in the opposing strand, or, an unlocked nucleic acid (UNA). 
     
     
         6 . The dsRNA molecule according to  claim 1 , wherein the thermally destabilizing modification of the duplex is located at position 6 or 7 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         7 . The dsRNA molecule according to  claim 1 , wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 14 and 16, counting from the 5′-end of the antisense strand. 
     
     
         8 . The dsRNA molecule according to  claim 1 , wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 6, 14 and 16, counting from the 5′-end of the antisense strand. 
     
     
         9 . The dsRNA molecule according to  claim 1 , wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5′-end of the antisense strand. 
     
     
         10 . The dsRNA molecule according to  claim 1 , comprising at least one ligand conjugated to the sense strand. 
     
     
         11 . The dsRNA molecule according to  claim 10 , wherein the at least one ligand comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide. 
     
     
         12 . The dsRNA molecule according to  claim 10 , wherein a ligand and is attached at the 5′-end or 3′-end of the sense strand. 
     
     
         13 . The dsRNA molecule according to  claim 10 , wherein a ligand is attached at an internal position of the sense strand. 
     
     
         14 . The dsRNA molecule according to  claim 10 , wherein a ligand comprises one or more GalNAc molecules attached through a bivalent or trivalent branched linker. 
     
     
         15 . The dsRNA molecule of  claim 10 , wherein a ligand is: 
       
         
           
           
               
               
           
         
       
       . 
     
     
         16 . The dsRNA molecule according to  claim 10 , wherein a ligand comprises a lipid. 
     
     
         17 . The dsRNA molecule according to  claim 10 , wherein a ligand comprises a cell targeting peptide. 
     
     
         18 . The dsRNA molecule according to  claim 10 , wherein a ligand improves nuclease resistance of the dsRNA molecule. 
     
     
         19 . The dsRNA molecule according to  claim 1 , wherein the dsRNA comprises at least four 2′-fluoro modifications or each of the sense and antisense strands comprise at least two 2′-fluoro modifications. 
     
     
         20 . The dsRNA molecule of  claim 1 , wherein the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications. 
     
     
         21 . The dsRNA molecule of  claim 20 , wherein the sense strand has 2′-fluoro modifications at positions complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         22 . The dsRNA molecule of  claim 20 , wherein the sense strand has 2′-fluoro modifications at positions complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         23 . The dsRNA molecule of  claim 1 , 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. 
     
     
         24 . The dsRNA molecule of  claim 23 , wherein the terminal three nucleotides at one end or both ends of the antisense strand are linked through phosphorothioate internucleotide linkages. 
     
     
         25 . The dsRNA molecule of  claim 1 , wherein the sense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages. 
     
     
         26 . The dsRNA molecule of  claim 25 , 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. 
     
     
         27 . The dsRNA molecule of  claim 26 , wherein the terminal three nucleotides at one end or both ends of the sense strand are linked through phosphorothioate internucleotide linkages. 
     
     
         28 . The dsRNA molecule of  claim 1 , having a blunt end at 5′end of the antisense strand. 
     
     
         29 . The dsRNA molecule of  claim 28 , having a blunt end at 3′end of the antisense strand. 
     
     
         30 . The dsRNA molecule of  claim 28 , having an at least two nucleotide overhang at 3′end of the antisense strand. 
     
     
         31 . The dsRNA molecule according to  claim 1 , wherein the duplex region is 19-23 base pairs in length. 
     
     
         32 . The dsRNA molecule according to  claim 1 , wherein the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides. 
     
     
         33 . The dsRNA molecule of  claim 1 , wherein the first base pair of the duplex region from the 5′ - end of the antisense strand is an AU base pair. 
     
     
         34 . The dsRNA molecule of  claim 1 , wherein the antisense strand comprises a 5′-phosphate or phosphoryl analog. 
     
     
         35 . The dsRNA molecule of  claim 34 , wherein the antisense strand comprises a 5′-vinyl phopshonate. 
     
     
         36 . The dsRNA molecule of  claim 1 , having no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         37 . The dsRNA molecule of  claim 1 , wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 14, and 16; and the thermally destabilizing modification of the duplex at position 6 or 7, each counting from the 5′ end of the antisense strand. 
     
     
         38 . The dsRNA molecule of  claim 37 , wherein the sense strand has 2′-fluoro modifications at positions complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. 
     
     
         39 . The dsRNA molecule of  claim 38 , wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 6, 14 and 16, counting from the 5′-end of the antisense strand. 
     
     
         40 . The dsRNA molecule of  claim 38 , wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5′-end of the antisense strand.

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