Methods and Compositions for Modulating Gene Expression Using Oligonucleotide Based Drugs Administered in vivo or in vitro

Abstract

Compositions and methods for down modulating target gene expression with RNA interference, as well as methods for administering said compositions are disclosed. The method comprises administering a first strand to a cell, incubating the cell for a time period suitable for uptake of the first oligo prior to administering a second strand, wherein the first strand and said second strand form an intracellular duplex which is effective to catalyze degradation of gene target mRNA or inhibit translation of said mRNA.

1 - 65 . (canceled) 
     
     
         66 . A formulation which inhibits expression and/or function of at least one target ribonucleic acid of interest in a cell within a subject comprising;
 a) a first composition comprising a first nucleic acid strand in a pharmaceutically acceptable vehicle for in vivo administration, said strand comprising modifications effective to promote stability and activity of said first strand in target tissue in vivo;   b) a second composition comprising a second nucleic acid strand in a pharmaceutically acceptable vehicle, said strand comprising modifications effective to promote stability and activity of said second strand in target tissue vivo;   wherein said compositions lack a pro-drug design or a carrier and at least one strand is complementary to the target ribonucleic acid of interest with no more than 4 mismatches with the target and comprising at least 14 contiguous-complementary nucleosides in the central region, the strands of a) and b) being complementary, capable of duplex formation, and between 16 and 24 nucleotides in length;   wherein said modifications are effective to
 i) increase the nuclease resistance of said strands in vivo to a half life greater than two hours in biological fluids; and 
 ii) alter the Tm of the duplex 
   wherein following sequential administration of said first and second strands in vivo, said strands exhibit increased inhibition of expression of said target ribonucleic acid sequence within said cell in a subject when compared to inhibition obtained using
 iii) formulations comprising duplexes of the strands of a) and b); or 
 iv) formulations comprising one of the single strands of a) or b) or 
 v) formulations comprising a double stranded siRNA directed to the same gene target 
   wherein the formulations of iii, iv, and v lack said modifications.   
     
     
         67 . The formulation of  claim 66 , wherein said strands comprise at least one modified backbone linkage selected from the group consisting of phosphorothioate linkages, methylphosphonate linkages, ethylphosphonate linkages, boranophosphate linkages, sulfonamide, carbonylamide, phosphorodiamidate, phosphorodiamidate linkages comprising a positively charged side group, phosphorodithioates, aminoethylglycine, phosphotriesters, aminoalkylphosphotriesters; 3′-alkylene phosphonates; 5′-alkylene phosphonates, chiral phosphonates, phosphinates, 3′-amino phosphoramidate, aminoalkylphosphoramidates, thionophosphoramidates; thionoalkyl-phosphonates, thionoalkylphosphotriesters, selenophosphates, 2-5′ linked boranophosphonate analogs, linkages having inverted polarity, abasic linkages, short chain alkyl linkages, cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, short chain heteroatomic or heterocyclic internucleoside linkages with siloxane backbones, sulfide, sulfoxide, sulfone, formacetyl linkages, thioformacetyl linkages, methylene formacetyl linkages, thioformacetyl linkages, riboacetyl linkages, alkene linkages, sulfamate backbones, methyleneimino linkages, methylenehydrazino linkages, sulfonate linkages, and amide linkages. 
     
     
         68 . The formulation of  claim 66 , wherein said strands comprise at least one modified sugar selected from the group consisting of 2′ fluoro, 2′-fluoro-D-arabinonucleic acid (FANA), 2′-O-methoxyethyl, 2′-O-methyl, a morpholino, a piperazine, and a locked nucleic acid (LNA). 
     
     
         69 . The formulation of  claim 68 , wherein said strands have alternating 2′-O-methyl modification and 2′fluoro modification. 
     
     
         70 . The formulation of  claim 68  wherein said strands have a mixture of phosphodiester and phosphorothioate backbone linkages. 
     
     
         71 . The formulation of  claim 66 , wherein said strands, when hybridized, comprise one or two 3′ overhangs, a 5′ overhang or a 3′ and 5′ overhang. 
     
     
         72 . The formulation of  claim 66 , wherein said strands form a blunt ended duplex. 
     
     
         73 . The formulation of  claim 66 , wherein one of said strands is a passenger strand which lacks a central linkage such that it occurs as two continuous oligos which hybridize with the complementary strand. 
     
     
         74 - 103 . (canceled) 
     
     
         104 . An in vitro method of identifying a formulation having an improved RNAi effect in vivo against a target gene, said method comprising;
 (i) obtaining a first and a second oligonucleotide sequence capable of forming a duplex;   (ii) adapting said first and/or said second oligonucleotide sequence to increase its nuclease resistance in vivo;   (iii) contacting said first oligonucleotide sequence with a cell expressing the target gene in vitro;   (iv) following step (iii) contacting said second oligonucleotide sequence with said cell of iii) and;   (v) determining whether the adaptations of step ii) enhance the RNAi effect of said formulation against said target gene as compared to the RNAi effect of the formulation on expression of the target gene without said adaptations, thereby identifying adaptations that enhance said RNAi effect.   
     
     
         105 . An in vitro method according to  claim 104  wherein said first oligonucleotide is truncated with respect to the second oligonucleotide. 
     
     
         106 . An in vitro method according to  claim 104  which are designed to create an overhang at one or both 3′ and/or a 5′ end when the duplex is formed intracellularly. 
     
     
         107 . An in vitro method according to  claim 104  wherein two or more first oligonucleotides are provided as a contiguous sequence. 
     
     
         108 . An in vitro method according to  claim 104  wherein said strands are selected from the group consisting of SEQ ID NOS: 119 to 377. 
     
     
         109 . A method for the treatment of a disorder listed in Table 2, comprising administration of at least one oligonucleotide selected from the group consisting of SEQ ID NOS: 119-377. 
     
     
         110 - 113 . (canceled)