Chimeric oligomeric compounds comprising alternating regions of northern and southern conformational geometry

Abstract

The present invention relates to novel chimeric oligomeric compounds having a plurality of alternating regions having either RNA like having northern or 3′-endo conformational geometry (3′-endo regions) or DNA like having southern or C2′-endo/O4′-endo conformational geometry. The oligomeric compounds of the present invention have shown reduction in mRNA levels in multiple in vitro and in vivo assay systems and are useful, for example, for investigative and therapeutic purposes.

1 . A chimeric oligomeric compound comprising from about 5 to about 80 linked nucleosides, wherein the chimeric oligomeric compound is divided into at least 5 separate regions, wherein each of the regions is a continuous sequence from 1 to about 5 nucleosides each comprising a 3′-endo sugar conformational geometry or a continuous sequence of from 1 to about 5 2′-deoxyribonucleosides, and wherein each of the regions comprising from 1 to about 5 2′-deoxyribonucleosides is internally located between two of the regions comprising 1 to about 5 nucleosides each comprising a 3′-endo sugar conformational geometry or at one of the 3′ or 5′-termini.  
     
     
         2 . The compound of  claim 1  comprising 5 separate regions.  
     
     
         3 . The compound of  claim 1  comprising 7 separate regions.  
     
     
         4 . The compound of  claim 1  comprising 9 separate regions.  
     
     
         5 . The compound of  claim 1  comprising 11 separate regions.  
     
     
         6 . The compound of  claim 1  comprising 13 separate regions.  
     
     
         7 . The compound of  claim 1  comprising 15 separate regions.  
     
     
         8 . The compound of  claim 1  comprising 17 separate regions.  
     
     
         9 . The compound of  claim 1  wherein each of the regions is from 1 to 4 nucleosides in length.  
     
     
         10 . The compound of  claim 1  wherein each of the regions is, independently, from 2 to 4 nucleosides in length.  
     
     
         11 . The compound of  claim 1  wherein each of the regions is, independently, from 1 to 3 nucleosides in length.  
     
     
         12 . The compound of  claim 1  wherein each of the regions is, independently, from 2 to 3 nucleosides in length.  
     
     
         13 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of nucleosides each comprising a 3′-endo sugar conformational geometry is, independently, from 1 to 4 nucleosides in length.  
     
     
         14 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of nucleosides each comprising a 3′-endo sugar conformational geometry is, independently, from 2 to 4 nucleosides in length.  
     
     
         15 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of nucleosides each comprising a 3′-endo sugar conformational geometry is, independently, from 3 to 4 nucleosides in length.  
     
     
         16 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of nucleosides each comprising a 3′-endo sugar conformational geometry is, independently, from 2 to 3 nucleosides in length.  
     
     
         17 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of 2′-deoxyribonucleosides is, independently, from 1 to 4 nucleosides in length.  
     
     
         18 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of 2′-deoxyribonucleosides is, independently, from 2 to 4 nucleosides in length.  
     
     
         19 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of 2′-deoxyribonucleosides is, independently, from 3 to 4 nucleosides in length.  
     
     
         20 . The compound of  claim 1  wherein each of the regions comprising a continuous sequence of 2′-deoxyribonucleosides is, independently, from 2 to 3 nucleosides in length.  
     
     
         21 . The compound of  claim 1  wherein each of the regions positioned at the terminal 3′ and 5′ ends comprise from 2 to 5 nucleosides in length.  
     
     
         22 . The compound of  claim 1  wherein each of the regions positioned at the terminal 3′ and 5′ ends comprise from 2 to 4 nucleosides in length.  
     
     
         23 . The compound of  claim 1  wherein each of the regions positioned at the terminal 3′ and 5′ ends comprise from 3 to 4 nucleosides in length.  
     
     
         24 . The compound of  claim 1  wherein each of the regions positioned at the terminal 3′ and 5′ ends comprise from 2 to 3 nucleosides in length.  
     
     
         25 . The compound of  claim 1  wherein each of the nucleosides comprising a 3′-endo sugar conformational geometry is, independently, a sugar modified nucleoside, a base modified nucleoside, or a nucleoside having one or more modifications that include both the base and the sugar.  
     
     
         26 . The compound of  claim 25  wherein each of the nucleosides comprising a 3′-endo sugar conformational geometry is a sugar modified nucleoside.  
     
     
         27 . The compound of  claim 26  wherein each of the sugar modified nucleosides, independently, comprises a 2′-substituent group.  
     
     
         28 . The compound of  claim 27  wherein each of the 2′-substituent groups is, independently, C 1 -C 20  alkyl, C 2 -C 20  alkenyl, C 2 -C 20  alkynyl, C 5 -C 20  aryl, —O-alkyl, —O-alkenyl, —O-alkynyl, —O-alkylamino, —O-alkylalkoxy, —O-alkylaminoalkyl, —O-alkyl imidazole, —OH, —SH, —S-alkyl, —S-alkenyl, —S-alkynyl, —N(H)-alkyl, —N(H)-alkenyl, —N(H)-alkynyl, —N(alkyl) 2 , —O-aryl, —S-aryl, —NH-aryl, —O-aralkyl, —S-aralkyl, —N(H)-aralkyl, phthalimido (attached at N), halogen, amino, keto (—C(═O)—R a ), carboxyl (—C(═O)OH), nitro (—NO 2 ), nitroso (—N═O), cyano (—CN), trifluoromethyl (—CF 3 ), trifluoromethoxy (—O—CF 3 ), imidazole, azido (—N 3 ), hydrazino (—N(H)—NH 2 ), aminooxy (—O—NH 2 ), isocyanato (—N═C═O), sulfoxide (—S(═O)—R a ), sulfone (—S(═O) 2 —R a ), disulfide (—S—S—R a ), silyl, heterocyclyl, carbocyclyl, an intercalator, a reporter group, a conjugate group, polyamine, polyamide, polyalkylene glycol, or a polyether of the formula (—O-alkyl)m a ; 
 wherein each R a  is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl, wherein the substituent group is haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy or aryl as well as halogen, hydroxyl, amino, azido, carboxy, cyano, nitro, mercapto, a sulfide group, a sulfonyl group, or a sulfoxide group;    or each sugar substituent group has one of formula I a  or II a :                          wherein:    R b  is O, S or NH;    R d  is a single bond, O, S or C(═O);    R e  is C 1 -C 10  alkyl, N(R k )(R m ), N(R k )(R n ), N═C(R p )(R q ), N═C(R p )(R r ), or has formula III a ;                          R p  and R q  are each independently hydrogen or C 1 -C 10  alkyl;    R r  is —R x —R y ;    each R s , R t , R u  and R v  is, independently, hydrogen, C(O)R w , 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 group is hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, or alkynyl;    or optionally, R u  and R v , together form a phthalimido moiety with the nitrogen atom to which they are attached;    each R w  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 k  is hydrogen, a nitrogen protecting group or —R x —R y ;    R p  is hydrogen, a nitrogen protecting group or —R x —R y ;    R x  is a bond or a linking moiety;    R y  is a chemical functional group, a conjugate group or a solid support medium;    each R m  and R n  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 the substituent group is hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl, NH 3   +, N(R   u )(R v ), guanidine, or acyl where the acyl is an acid amide or an ester;    or R m  and R n , together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group;    R i  is OR z , SR z , or N(R z ) 2 ;    each R z  is, independently, H, C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C(═NH)N(H)R u , C(═O)N(H)R u , or OC(═O)N(H)R u ;    R f , R g  and R h  comprise a ring system comprising from about 4 to about 7 carbon atoms or comprising from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein the heteroatoms are oxygen, nitrogen, or sulfur and wherein the ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;    R j  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 k )(R m ) OR k , halo, SR k  or CN;    m a  is 1 to about 10;    each mb is, independently, 0 or 1;    mc is 0 or an integer from 1 to 10;    md is an integer from 1 to 10;    me is from 0, 1 or 2; and    provided that when mc is 0, md is greater than 1.    
     
     
         29 . The compound of  claim 26  wherein each of the 2′-substituent groups is, independently, O(CH 2 ) 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 ON(CH 3 ) 2 , O(CH 2 ) 2 N(CH 3 ) 2 , OCH 2 C(═O)N(H)CH 3 , OCH 3 , O(CH 2 ) 2 NH 2 , O(CH 2 ) 2 N(CH 3 ) 2 , O(CH 2 ) 3 NH 2 , O(CH 2 ) 3 N(H)CH 3 , CH 2 CH═CH 2 , or O(CH 2 ) 2 S(O)CH 3 .  
     
     
         30 . The compound of  claim 25  wherein each of the 2′-substituent groups is, independently, OCH 3 , OCH 2 CH 2 OCH 3 , N 3 , CH 2 CHCH 2 , C 1 -C 20  alkyl, COOH, CONR 1 R 2 , CONR 1 R 2 , NR 1 R 2 , —SR 1 , NR 1 OR 2 , or F wherein each R 1  and R 2  is, independently, hydrogen, 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 the substituent group is hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl, guanidine, or acyl where the acyl is an acid amide or an ester.  
     
     
         31 . The compound of  claim 1  wherein at least one of the nucleosides of one of the regions comprising 3′-endo sugar conformational geometry is a xlyo nucleoside.  
     
     
         32 . The compound of  claim 1  wherein at least one of the nucleosides of one of the regions comprising 3′-endo sugar conformational geometry is an arbino nucleoside.  
     
     
         33 . The compound of  claim 1  wherein at least one of the nucleosides of one of the regions comprising 3′-endo sugar conformational geometry has a bicyclic sugar moiety.  
     
     
         34 . The compound of  claim 33  wherein at least one of the bicyclic sugar moieties is a locked nucleic acid (LNA).  
     
     
         35 . The compound of  claim 23  wherein at least one of the nucleosides comprising a 3′-endo sugar conformational geometry is a base modified nucleoside.  
     
     
         36 . The compound of  claim 35  wherein the modified base nucleoside is 5-methyl cytosine.  
     
     
         37 . The compound of  claim 35  wherein each cytosine containing nucleoside is substituted with a 5-methyl cytosine containing nucleoside.  
     
     
         38 . The compound of  claim 35  wherein at least one of the nucleosides of one of the regions comprising 3′-endo sugar conformational geometry comprises a modified heterocyclic base moiety selected from the group consisting of 2-thiothymine, 2′-O-methylpseudouricyl, 7-halo-7-deaza purine, 7-propyne-7-deaza purine, and 2,6-diaminopurine.  
     
     
         39 . The compound of  claim 1  wherein each of the nucleosides is linked by an internucleoside linking group independently selected from the group consisting of phosphodiester, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 5′-alkylene phosphonate, chiral phosphonate, phosphinate, phosphoramidate, 3′-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, and boranophosphate.  
     
     
         40 . The compound of  claim 39  wherein each of the nucleosides is, independently, linked by phosphodiester or phosphorothioate.  
     
     
         41 . The compound of  claim 40  wherein each of the nucleosides is linked by a phosphorothioate internucleoside linking group.  
     
     
         42 . The compound of  claim 1  wherein each nucleoside of the regions comprising 3′-endo conformational geometry comprises a 2′-O—CH 2 CH 2 —O—CH 3  group.  
     
     
         43 . The compound of  claim 1  comprising from about 5 to 50 nucleosides in length.  
     
     
         44 . The compound of  claim 1  comprising from about 12 to 30 nucleosides in length.  
     
     
         45 . The compound of  claim 1  comprising from about 15 to 25 nucleosides in length.  
     
     
         46 . The compound of  claim 1  comprising from about 21 to 25 nucleosides in length.  
     
     
         47 . The compound of  claim 1  wherein each nucleoside of the regions comprising 3′-endo conformational geometry comprises a 2′-O—CH 3  group.  
     
     
         48 . The compound of  claim 1  wherein each nucleoside of the regions comprising 3′-endo conformational geometry comprises a 2′-fluoro group.  
     
     
         49 . The compound of  claim 1  wherein each nucleoside of the regions comprising 3′-endo conformational geometry is a LNA nucleoside.  
     
     
         50 . The compound of  claim 1  comprising from about 13 to 30 nucleosides in length.  
     
     
         51 . A method of reducing target mRNA levels in a cell in vitro comprising contacting the cell with a gap-disabled compound listed in Table 13 or Table 26.  
     
     
         52 . A method of reducing cell surface expression of CD86 in an MH-S cell comprising contacting the cell with a gap-disabled compound.  
     
     
         53 . A method of reducing viability of a cell comprising contacting the cell with a gap-disabled compound listed in Table 28.  
     
     
         54 . An oligomeric compound comprising the gap-disabled motif  2 -1- 1 -2- 1 -1- 1 -1- 1 -1- 1 -1- 1 -3- 2 .  
     
     
         55 . A method of reducing the hepatotoxicity of an oligonucleotide comprising incorporating a gap-disabled motif into the oligonucleotide.  
     
     
         56 . The method of  claim 55  wherein the gap-disabled motif is  2 -1- 1 -2-   1-   1-   1-   1-   1-   1-   1-   1- 1 -3- 2  or  3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3 .  
     
     
         57 . The method of  claim 55  wherein the gap-disabled motif comprises at least 9 alternating 3′-endo and 2′-endo regions.  
     
     
         58 . A double-stranded heteroduplex compound comprising a gap-disabled oligonucleotide having the gap-disabled motif of  3 -2- 1 -3- 1 -3- 1 -3- 3 .  
     
     
         59 . A method of eliciting cleavage of a target RNA comprising contacting the target RNA with a gap-disabled compound comprising the gap-disabled motif of  3 -2- 1 -3- 1 -3- 1 -3- 3 .  
     
     
         60 . The method of  claim 59  wherein the cleavage of the target RNA occurs in the nucleus.  
     
     
         61 . The method of  claim 59  wherein the cleavage position on the target RNA occurs within the 2′-deoxynucleotide gaps.  
     
     
         62 . The method of  claim 59  wherein the cleavage occurs at a guanine residue.  
     
     
         63 . A method of reducing target RNA levels in an animal comprising contacting the animal with a gap-disabled compound comprising a gap-disabled motif listed in Table 13 or Table 26 and wherein the gap-disabled compound comprises a nucleobase sequence substantially complementary to a portion of the target RNA.  
     
     
         64 . A method of lowering cholesterol or triglycerides in an animal comprising contacting the animal with a gap-disabled compound comprising the gap-disabled motif  3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3 .  
     
     
         65 . A method of lowering plasma leptin, glucose, or plasma insulin in an animal comprising contacting the animal with a gap-disabled compound having the gap-disabled motif  3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3 .  
     
     
         66 . A method of lowering body weight, fat depot weight or food intake in an animal comprising contacting the animal with a gap-disabled compound comprising the gap-disabled motif  3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3 .  
     
     
         67 . A method of reducing serum cholesterol, triglycerides or body weight in an obese animal comprising contacting the animal with a gap-disabled compound comprising the gap-disabled motif of  3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3 .