Oligonucleotide Conjugates

Abstract

The invention relates to the field of oligonucleotide therapeutics, and in particular to the use of a cleavable, e.g. a phosphodiester region covalently attached to a conjugate, a targeting group or blocking group to enhance the properties of the oligonucleotides, for example to improve the therapeutic index.

1 .- 52 . (canceled) 
     
     
         53 . A method of synthesizing an oligomeric compound 8-35 nucleotides in length, comprising three regions:
 a first region (region A), which is an antisense oligomer comprising 7-26 contiguous nucleotides complementary to a nucleic acid target, wherein the first region comprises at least one nucleoside analog and wherein the internucleoside linkages of the first region comprise at least 50% internucleoside linkages other than phosphodiester;   a second region (region B) which comprises between 1-10 nucleotides, which is covalently linked to the 5′ or 3′ nucleotide of the first region by an internucleoside linkage, wherein either:
 (i) the internucleoside linkage between the first and second region is a phosphodiester linkage and the nucleoside of the second region adjacent to the first region is either DNA or RNA; and/or 
 (ii) at least 2 nucleosides of the second region are phosphodiester linked DNA or RNA nucleosides; 
   a third region which comprises a conjugate moiety, a targeting moiety, or a blocking moiety, wherein the third region is covalently linked to the second region,   
       said method comprising 
       either:
 (a) a step of providing an oligonucleotide synthesis support to which is attached a moiety selected from the group consisting of: (i) a linker group (—Y—), (ii) a group (X—) selected from the group consisting of a conjugate, a targeting group and a blocking group, and (iii) a —Y—X group; and 
 (b) a step of oligonucleotide synthesis of region B followed by region A, 
 
       or:
 (a′) a step of oligonucleotide synthesis of a first region (A) and a second region (B), wherein the first region (A) is attached to an oligonucleotide synthesis support and the synthesis step is followed by 
 (b′) a step of adding a moiety selected from the group consisting of: (i) a linker group (—Y —); (ii) a group (X—) selected from the group consisting of a conjugate, a targeting group or a blocking group, and (iii) an —Y—X group; 
 
       followed by
 (c) the cleavage of the oligomeric compound from the support, wherein, if the moiety is a linker group (Y), said method further comprises, 
 (d) a further step of adding a conjugate, a blocking, or targeting group to the linker group (Y), wherein said step is performed either prior to or subsequent to cleavage of the oligomeric compound from the oligonucleotide synthesis support. 
 
     
     
         54 . The method of  claim 53 , comprising steps (a), (b) and (c). 
     
     
         55 . The method of  claim 53 , comprising steps (a′), (b′) and (e) 
     
     
         56 . A method of synthesizing an oligomeric compound of 8-35 nucleotides in length, comprising three regions:
 a first region (region A), which is an antisense oligomer of 7-26 contiguous nucleotides complementary to a nucleic acid target, wherein the first region comprises at least four nucleoside analogues and wherein the internucleoside linkages of the first region are internucleoside linkages other than phosphodiester;   a second region (region B) which is covalently linked to the 5′ or 3′ nucleotide of the first region via a phosphodiester internucleoside linkage, wherein the second region consists of 2-10 phosphodiester linked DNA or RNA nucleosides; and   a third region which comprises a conjugate moiety or a targeting moiety wherein the third region is covalently linked to the second region,   
       said method comprising 
       either:
 (a) a step of providing an oligonucleotide synthesis support to which is attached a moiety selected from the group consisting of: (i) a linker group (—Y—), (ii) a group (X—) selected from the group consisting of a conjugate and a targeting group, and (iii) a —Y—X group; and 
 (b) a step of oligonucleotide synthesis of region B followed by region A, 
 
       or:
 (a′) a step of oligonucleotide synthesis of a first region (A) and a second region (B), wherein the first region (A) is attached to an oligonucleotide synthesis support and the synthesis step is followed by 
 (b′) a step of adding a moiety selected from the group consisting of: (i) a linker group (—Y—); (ii) a group (X—) selected from the group consisting of a conjugate and a targeting group, and (iii) a —Y—X group, 
 
       followed by
 (c) the cleavage of the oligomeric compound from the support, wherein, if the moiety is a linker group (Y), said method further comprises 
 (d) a further step of adding a conjugate or a targeting group to the linker group (Y), wherein said further step is performed either prior to or subsequent to cleavage of the oligomeric compound from the oligonucleotide synthesis support. 
 
     
     
         57 . The method of  claim 56 , comprising steps (a), (b) and (e). 
     
     
         58 . The method of  claim 56 , comprising steps (a′), (b′) and (e). 
     
     
         59 . The method according to  claim 56 , wherein the internucleoside linkages other than phosphodiester in the first region of the oligomeric compound are selected from the group consisting of phosphorothioate linkages, phosphorodithioate linkages and boranophosphate linkages. 
     
     
         60 . The method according to  claim 56 , wherein the first region is a gapmer, a mixmer, or a totalmer. 
     
     
         61 . The method according to  claim 56 , wherein the first region comprises at least one bicyclic nucleotide analogue (LNA). 
     
     
         62 . The method according to  claim 56 , wherein the second region is covalently linked to the third region at the terminal nucleoside of the second region. 
     
     
         63 . The method according to  claim 56 , wherein the second and third regions are covalently joined by a linker group. 
     
     
         64 . The method according to  claim 56 , wherein the linkage between the second and third regions comprises a group selected from phosphodiester, a phosphorothioate, a phosphorodithioate and a boranophosphate group. 
     
     
         65 . The method according to  claim 56 , wherein the third region comprises a non-nucleotide moiety selected from a sterol and a carbohydrate. 
     
     
         66 . The method according to claim  13 , wherein the third region comprises cholesterol. 
     
     
         67 . The method according to claim  13 , wherein the third region comprises a GalNac cluster. 
     
     
         68 . The method according to  claim 56 , wherein the third region comprises a moiety selected from the group consisting of: a lipophilic group, a protein, a peptide, an antibody or fragment thereof, a polymer, a reporter group, a dye, a receptor ligand, a small molecule drug, a prodrug, and a vitamin. 
     
     
         69 . A method of synthesizing an oligomeric compound composed of three regions:
 a first region (region A), which is an antisense oligomer of 10-22 contiguous nucleotides complementary to a nucleic acid target, wherein the first region is a gapmer;   a second region (region B) which comprises between 1-10 nucleotides, which is covalently linked to the 5′ or 3′ nucleotide of the first region, wherein: (i) the internucleoside linkage between the first and second region is a phosphodiester linkage and the nucleoside of the second region adjacent to the first region is either a DNA nucleotide or an RNA nucleotide; and/or (ii) at least one nucleotide of the second region is a phosphodiester linked DNA nucleotide or RNA nucleotide; and   a third region which comprises a conjugate moiety or a targeting moiety, wherein the third region is covalent linked to the 5′ or 3′ terminus of the second region,   
       said method comprising 
       either:
 (a) a step of providing an oligonucleotide synthesis support to which is attached a moiety selected from the group consisting of: (i) a linker group (—Y—); (ii) a group (X—) selected from the group consisting of a conjugate and a targeting group, and (iii) a —Y—X group 
 
       and
 (b) a step of oligonucleotide synthesis of region B followed by region A, 
 
       or:
 (a′) a step of oligonucleotide synthesis of a first region (A) and a second region (B), wherein the first region (A) is attached to an oligonucleotide synthesis support and the synthesis step is followed by 
 (b′) a step of adding a moiety selected from the group consisting of: (i) a linker group (—Y—); (ii) a group (X—) selected from the group consisting of a conjugate and a targeting group, and (iii) a —Y—X group, 
 
       followed by
 (c) the cleavage of the oligomeric compound from the support wherein, if the moiety is a linker group (Y), said method further comprises 
 (d) a further step of adding a conjugate or a targeting group to the linker group (Y), wherein said further step is performed either prior to or subsequent to cleavage of the oligomeric compound from the oligonucleotide synthesis support. 
 
     
     
         70 . The method of  claim 69 , comprising steps (a), (b) and (c). 
     
     
         71 . The method of  claim 69 , comprising steps (a′), (b′) and (c). 
     
     
         72 . The method according to  claim 69 , wherein the internucleoside linkages of the first region comprise at least one internucleoside linkage other than phosphodiester. 
     
     
         73 . The method according to  claim 72 , wherein the internucleoside linkages other than phosphodiester in the first region of the oligomeric compound are selected from the group consisting of phosphorothioate, phosphorodithioate and boranophosphate. 
     
     
         74 . The method according to  claim 73 , wherein at least 75%, of the internucleoside linkages of the first region are other than phosphodiester. 
     
     
         75 . The method according to  claim 73 , wherein all of the internucleoside linkages of the first region are other than phosphodiester. 
     
     
         76 . The method according to  claim 69 , wherein the first region of the oligomeric compound comprises at least one bicyclic nucleotide analogue (LNA). 
     
     
         77 . The method of  claim 68 , wherein the first region of the oligomeric compound is covalently linked to the second region via a phosphodiester linkage. 
     
     
         78 . The method according to  claim 69 , wherein the second region of the oligomeric compound is 1 to 4 nucleotides long. 
     
     
         79 . The method according to  claim 69 , wherein the second region consists of one DNA nucleotide or one RNA nucleotide. 
     
     
         80 . The method according to  claim 69 , wherein the second region of the oligomeric compound comprises at least two nucleotides selected from the group of DNA nucleotides and RNA nucleotides, wherein the nucleotides are linked by phosphodiester linkages. 
     
     
         81 . The method according to  claim 69 , wherein the second region of the oligomeric compound comprises at least two nucleotides and the linkage between the first two nucleotide adjacent to the first region is a phosphodiester linkage. 
     
     
         82 . The method according to  claim 69 , wherein the second region of the oligomeric compound is covalently linked to the third region at the terminal nucleoside of the second region. 
     
     
         83 . The method according to  claim 81 , wherein the third region of the oligomeric compound is covalently linked to the second region via a phosphodiester linkage. 
     
     
         84 . The method according to  claim 69 , wherein the first region and the second region of the oligomeric compound are covalently linked by a phosphodiester linkage and the second region and the third region are covalently linked by a phosphodiester linkage. 
     
     
         85 . The method according to  claim 69 , wherein the third region of the oligomeric compound comprises cholesterol. 
     
     
         86 . The method according to  claim 69 , wherein the third region of the oligomeric compound comprises a GalNac cluster.