Glycoconjugates of rna interference agents

Abstract

The present invention relates to agents, compositions and methods for inhibiting the expression of a target gene, comprising an RNAi agent bearing at least one galactosyl moiety. These are useful for delivering the gene expression inhibiting activity to cells, particularly hepatocytes, and more particularly in therapeutic applications.

1 - 20 . (canceled) 
     
     
         21 . An RNAi agent for inhibiting the expression of a target gene in a cell, wherein the RNAi agent consists essentially of two mutually complementary oligoribonucleotide strands of between 15 and 30 nucleotides in length, wherein an ASGPR ligand comprising at least two galactose moieties is conjugated to at least one of the oligoribonucleotide strands via a branched linker and via a phosphate or phosphate modification at the 5′ end, and wherein at least one oligoribonucleotide strand is complementary to at least one portion of a mRNA corresponding to the target gene. 
     
     
         22 . The RNAi agent of  claim 21 , wherein the ASGPR ligand conjugated to the oligoribonucleotide through the branched linker has a structure of formula (III) 
       
         
           
           
               
               
           
         
         wherein 
         Z 3  and Z 4  are independently O or S; and 
         the ASGPR ligand is linked to O via at least one linker group 
       
       
         
           
           
               
               
           
         
       
       wherein Z 1  and Z 2  are independently O or S, and n is 1-20. 
     
     
         23 . The RNAi agent of  claim 22 , wherein at least one of Z 3  and Z 4  is S. 
     
     
         24 . The RNAi agent of  claim 22 , wherein both of Z 3  and Z 4  are O. 
     
     
         25 . The RNAi agent of  claim 21 , wherein the ASGPR ligand is linked to the branched linker through an intervening linker. 
     
     
         26 . The RNAi agent of  claim 25 , wherein the ASGPR ligand linked to the branched linker through the intervening linker has a structure of formula (IV)
   ASGPR ligand-O—CH 2 CH 2 (OCH 2 CH 2 ) n OP(Z 5 )(Z 6 )-branched-linker   Formula (IV)
   wherein n is 1-20; and   Z 5  and Z 6  are each independently O or S.   
     
     
         27 . The RNAi agent of  claim 26 , wherein n is 3. 
     
     
         28 . The RNAi agent of  claim 26 , wherein at least one of Z 5  and Z 6  is S. 
     
     
         29 . The RNAi agent of  claim 26 , wherein both of Z 5  and Z 6  are O. 
     
     
         30 . The RNAi agent of  claim 21 , wherein the galactose moieties are N-acetylgalactosamine moieties. 
     
     
         31 . The RNAi agent of  claim 30 , wherein the distance between the N-acetylgalactosamine moieties is at least 4 Å. 
     
     
         32 . The RNAi agent of  claim 21 , wherein the 5′ end phosphate modification is characterized by replacing one or both non-bridging oxygen atoms of the phosphate by B, C, N, or a group containing thereof; S; Se; H; or OR, wherein R is alkyl or aryl. 
     
     
         33 . The RNAi agent of  claim 21 , wherein the 5′ end phosphate modification is characterized by replacing one or both bridging oxygen atoms of the phosphate by C, N, or a group containing thereof; or S. 
     
     
         34 . The RNAi agent of  claim 21 , wherein the 5′ end phosphate modification is selected from the group consisting of phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkylene or arylene phosphonates, and phosphotriesters. 
     
     
         35 . The RNAi agent of  claim 21 , wherein the RNAi agent is capable of inhibiting the expression of the target gene in the cell. 
     
     
         36 . The RNAi agent of  claim 35 , wherein the cell harbors an asialoglycoprotein receptor on its surface. 
     
     
         37 . The RNAi agent of  claim 36 , wherein the cell is a hepatocyte. 
     
     
         38 . A pharmaceutical composition, comprising (i) an RNAi agent of claim  1 ; and (ii) a pharmaceutically acceptable excipient. 
     
     
         39 . A method for the manufacture of an RNAi agent of claim  1 , comprising the steps of (i) synthesizing two mutually complementary oligoribonucleotide strands of between 15 and 30 nucleotides in length, wherein at least one of the oligoribonucleotides is coupled to a ligand comprising a linker group and at least one galactose moiety; and (ii) effecting the hybridization of the at least two mutually complementary oligoribonucleotides. 
     
     
         40 . The method of  claim 39 , further comprising the step of formulating the RNAi agent with a pharmaceutically acceptable excipient.