Human RNase H1 and oligonucleotide compositions thereof

Abstract

The present invention provides oligonucleotides that can serve as substrates for human Type 2 RNase H. The present invention is also directed to methods of using these oligonucleotides in enhancing antisense oligonucleotide therapies.

What is claimed is:  
     
         1 . A mixed sequence oligonucleotide comprising at least 12 nucleotides in length and having a 3′ end and a 5′ end and divided into a first portion and a further portion, 
 said first portion being capable of supporting cleavage of a complementary target RNA by human RNase H1 polypeptide,  
 said further portion being incapable of supporting said cleavage by said RNase H1;  
 wherein said first portion comprises at least 6 nucleotides and is positioned in said oligonucleotide such that at least one of said 6 nucleotides is 8 to 12 nucleotides from the 3′ end of said oligonucleotide.  
 
     
     
         2 . The oligonucleotide of  claim 1  comprising at least one CA nucleotide sequence within said first portion.  
     
     
         3 . The oligonucleotide of  claim 1  comprising from about 12 to about 50 nucleotides.  
     
     
         4 . The oligonucleotide of  claim 1  comprising from about 12 to about 25 nucleotides.  
     
     
         5 . The oligonucleotide of  claim 1  wherein each of said nucleotides of said first portion have B-form conformational geometry and are joined together in a continuous sequence.  
     
     
         6 . The oligonucleotide of  claim 1  wherein each of said nucleotides of said first portion is, independently, a 2′-deoxyribonucleotide, a 2′-SCH 3  ribonucleotide, a 2′-NH 2  ribonucleotide, a 2′-NH(C 1 -C 2  alkyl) ribonucleotide, a 2′-N(C 1 -C 2  alkyl) 2  ribonucleotide, a 2′-CF 3  ribonucleotide, a 2′=CH 2  ribonucleotide, a 2′=CHF ribonucleotide, a 2′=CF 1  ribonucleotide, a 2′-CH 3 ribonucleotide, a 2′-C 2 H 5  ribonucleotide, a 2′-CH=CH 2  ribonucleotide or a 2′-C≡CH ribonucleotide.  
     
     
         7 . The oligonuceotide of  claim 1  wherein each of said nucleotides of said first portion is a 2′-deoxyribonucleotide.  
     
     
         8 . The oligonucleotide of  claim 1  wherein each of said nucleotide of said first portion is, independently, a 2′-CN arabinonucleotide, a 2′-F arabinonucleotide, a 2′-Cl arabinonucleotide, a 2′-Br arabinonucleotide, a 2′-N 3  arabinonucleotide, a 2′-OH arabinonucleotide, a 2′-O-CH 3  arabinonucleotide or a 2′-dehydro-2′-CH 3  arabinonucleotide.  
     
     
         9 . The oligonucleotide of  claim 1  wherein each of said nucleotides of said first portion is, independently, a 2′-F arabinonucleotide, a 2′-OH arabinonucleotide or a 2′-O-CH 3  arabinonucleotide.  
     
     
         10 . The oligonucleotide of  claim 1  wherein each of said nucleotides of said first portion is, independently, a 2′-F arabinonucleotide or a 2′-OH arabinonucleotide.  
     
     
         11 . The oligonucleotide of  claim 1  wherein said nucleotides of said first portion are joined together in said continuous sequence by phosphate, phosphorothioate, phosphorodithioate or boranophosphate linkages.  
     
     
         12 . The oligonucleotide of  claim 1  wherein said further portion includes a plurality of nucleotides, at least some of said nucleotides comprise a 2′ substituent group wherein each substituent group is, independently, hydroxyl, C 1 -C 20  alkyl, C 2 -C 20  alkenyl, C 2 -C 20  alkynyl, halogen, amino, thiol, keto, carboxyl, nitro, nlitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkenyl, O-alkynyl, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether; 
 or each substituent group has one of formula I or II:  
                     
 wherein:  
 Z 0  is O, S or NH;  
 J is a single bond, O or C(═O);  
 E is C 1 -C 10  alkyl, N(R 1 ) (R 2 ), N(R 1 ) (R 5 ), N═C(R 1 ) (R 2 ), N═C(R 1 ) (R 5 ) or has one of formula III or IV;  
                     
 each R 6 , R 7 , R 8 , R 9  and R 10  is, independently, hydrogen, C(O)R 11 , substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;  
 or optionally, R 7  and R 8 , together form a phthalimido moiety with the nitrogen atom to which they are attached;  
 or optionally, R 9  and R 10 , together form a phthalimido moiety with the nitrogen atom to which they are attached;  
 each R 11  is, independently, substituted or unsubstituted C 1 -C 10  alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;  
 R 5  is T-L,  
 T is a bond or a linking moiety;  
 L is a chemical functional group, a conjugate group or a solid support material;  
 each R 1  and R 2  is, independently, H, a nitrogen protecting group, substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, wherein said substitution is OR 3 , SR 3 , NH 3   + , N (R 3 ) (R 4 ), guanidino or acyl where said acyl is an acid amide or an ester;  
 or R 1  and R 2 , together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;  
 or R 1 , T and L, together, are a chemical functional group;  
 each R 3  and R 4  is, independently, H, C 1 -C 10  alkyl, a nitrogen protecting group, or R 3  and R 4 , together, are a nitrogen protecting group;  
 or R 3  and R 4  are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;  
 Z 4  is OX, SX, or N(X) 2 ;  
 each X is, independently, H, C 1 -C 8  alkyl, C 1 -1-C 6  haloalkyl, C(═NH)N(H)R 5 , C(═O)N(H)R 5  or OC(═O)N(H)R 5 ;  
 R 5  is H or C 1 -C 8  alkyl;  
 Z 1 , Z 2  and Z 3  comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;  
 Z 5  is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R 1 ) (R 2 ) OR 1 , halo, SR 1  or CN;  
 each q 1  is, independently, an integer from 1 to 10;  
 each q 2  is, independently, 0 or 1;  
 q 3  is 0 or an integer from 1 to 10;  
 q 4  is an integer from 1 to 10; and  
 q 5  is from 0, 1 or 2;  
 provided that when q 3  is 0, q 4  is greater than 1.  
 
     
     
         13 . The oligonucleotide of  claim 1  wherein each of said nucleotides of said further portion is, independently, a 2′-F ribonucleotide, a 2′-O-(C 1 -C 6  alkyl) ribonucleotide, or a 2′-O-(C 1 -C 6  substituted alkyl) ribonucleotide wherein the substitution is C 1 -C 6  ether, C 1 -C 6  thioether, amino, amino(C 1 -C 6  alkyl) or amino(C 1 -C 6  alkyl) 2 .  
     
     
         14 . The oligonucleotide of  claim 1  wherein said nucleotides of said further portion are joined together in a continuous sequence by 3′-5′ phosphodiester, 2′-5′ phosphodiester, phosphorothioate, Sp phosphorothioate, Rp phosphorothioate, phosphorodithioate, 3′-deoxy-3′-amino phosphoroamidate, 3′-methylenephosphonate, methylene(methylimino), dimethylhydrazino, amide 3, amide 4 or boranophosphate linkages.  
     
     
         15 . The oligonucleotide of  claim 1  wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion.  
     
     
         16 . The oligonucleotide of  claim 1  wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         17 . The oligonucleotide of  claim 1  wherein at least two of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion and at least two of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         18 . The oligonucleotide of claimL 1 wherein at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion.  
     
     
         19 . The oligonucleotide of  claim 1  wherein at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         20 . The oligonucleotide of  claim 1  wherein at least four of said nucleotides of said further portion are joined together in a continuous sequence that is positioned 3′ to said first portion and at least four of said nucleotides or said further portion are joined together in a continuous sequence that is positioned 5′ to said first portion.  
     
     
         21 . A mixed sequence oligonucleotide comprising at least 8 nucleotides and having a CA nucleotide sequence wherein at least one of the two nucleotides of said CA sequence is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide.  
     
     
         22 . The oligonucleotide of  claim 21  wherein said oligonucleotide is capable of supporting cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         23 . A mixed sequence chimeric oligonucleotide comprising at least 8 nucleotides and having a CA nucleotide sequence wherein at least one of the two nucleotides of said CA sequence is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide.  
     
     
         24 . The chimeric oligonucleotide of  claim 23  wherein said oligonucleotide is capable of supporting cleavage of a complementary target RNA by human RNase H1 polypeptide.  
     
     
         25 . A mixed sequence oligonucleotide comprising 8 to 25 nucleotides and having a CA nucleotide sequence wherein at least one of the nucleotides of said CA sequence is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide.  
     
     
         26 . A mixed sequence chimeric oligonucleotide comprising 8 to 25 nucleotides and having a CA nucleotide sequence wherein at least one of the nucleotides of said CA sequence is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide.  
     
     
         27 . A chimeric oligonucleotide comprising 8 to 25 nucleotides and having a portion capable of supporting cleavage of a complementary target RNA by human RNase H1 polypeptide wherein said portion supporting said cleavage is at least 6 nucleotides in length and is positioned in said oligonucleotide such that at least one of said 6 nucleotides is positioned 8 to 12 nucleotides from the 3′ end of said oligonucleotide.  
     
     
         28 . The oligonucleotide of  claim 27  wherein said oligonucleotide comprises at least one CA nucleotide sequence within said portion supporting said cleavage.