Double-stranded ribonucleic acid with increased effectiveness in an organism

Abstract

The present invention relates to a method for the targeted selection of a double-stranded ribonucleic acid (dsRNA) consisting of two single strands that exhibits increased effectiveness in inhibiting the expression of a target gene by means of RNA interference, wherein at least end of the dsRNA comprises a nucleotide overhang of 1 to 4 unpaired nucleotides in length; wherein the unpaired nucleotide adjacent to the terminal nucleotide pair comprises a purine base; and wherein the terminal nucleotide pair on both ends of the dsRNA is a G-C pair, or at least two of the last four consecutive terminal nucleotide pairs are G-C pairs.

1 . A double-stranded ribonucleic acid (dsRNA) having improved stability in cells comprising a sense strand and an antisense strand, wherein the dsRNA comprises at least one chemically modified nucleotide, an antisense strand of less than 30 nucleotides, one blunt end and one single stranded overhang of less than or equal to 15 nucleotides in length; wherein a single-stranded overhang is located at the 3′-end of the antisense strand; and wherein the terminal base pair of the first double-stranded end comprises a guanine-cytosine (G-C) base pair or the four consecutive terminal base pairs of the first double-stranded end comprises at least two G-C base pairs; wherein the terminal base pair of the second double-stranded end comprises a guanine-cytosine (G-C) base pair or the four consecutive terminal base pairs of the second double-stranded end comprises at least two G-C base pairs. 
     
     
         2 . The dsRNA of  claim 1 , wherein each nucleotide overhang independently consists of 2 unpaired nucleotides. 
     
     
         3 . The dsRNA of  claim 1 , wherein the region of the antisense strand that is complementary to the target gene is 19 to 24 nucleotides in length. 
     
     
         4 . The dsRNA of  claim 1 , wherein the antisense strand is 20 to 28 nucleotides in length. 
     
     
         5 . The dsRNA of  claim 1 , wherein the antisense strand is 20 to 23 nucleotides in length. 
     
     
         6 . The dsRNA of  claim 1 , wherein the chemically modified nucleotide comprises a non-natural base. 
     
     
         7 . The dsRNA of  claim 1 , wherein the chemically modified nucleotide comprises a 2′ modification. 
     
     
         8 . The dsRNA of  claim 7 , wherein the 2′modification is selected from the group consisting of a 2′-amino modification, a 2′-O-methyl modification, a 2′-O-ethyl modification, a 2′-O-propyl modification, a 2′-O-allyl modification, a 2′-O-aminoalkyl modification, and a 2′-deoxy-2′-fluoro modification. 
     
     
         9 . The dsRNA of  claim 1 , wherein the chemically modified comprises modified internucleoside linkages or backbones. 
     
     
         10 . The dsRNA of  claim 9 , wherein the modified internucleoside is selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalklyphosphotriesters, and boranophosphates. 
     
     
         11 . The dsRNA of  claim 10 , wherein the internucleoside are modified in a normal 3′-5′ linkages, or 2′-5′ linked analogs. 
     
     
         12 . The dsRNA of  claim 10 , wherein the internucleoside are modified in an inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. 
     
     
         13 . The dsRNA of  claim 1 , wherein the dsRNA comprises 1 to 6 mismatches in the terminal regions of the 5′ and/or 3′ terminus. 
     
     
         14 . A pharmaceutical composition for inhibiting the expression of a target gene by means of RNA interference, comprising a dsRNA of  claim 1 , or a salt, prodrug or hydrate thereof; and a pharmaceutically acceptable carrier. 
     
     
         15 . A method for inhibiting the expression of a target gene in a cell, comprising:
 (a) introducing into the cell a dsRNA of  claim 1 , or a salt, prodrug or hydrate thereof; and   (b) maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of the target gene.   
     
     
         16 . The method of  claim 15 , wherein the cell is a mammalian cell. 
     
     
         17 . The method of  claim 15 , wherein the cell is a human cell. 
     
     
         18 . The method of  claim 15 , wherein the target gene is selected from the group consisting of 11-hyroxysteroid dehydrogenase-1, acetyl-CoA-carboxylase-2, acyl CoA: DAG acyltransferase-1, Adenosine A2 receptor, akt, AML-ETO, amyloid beta precursor protein (APP), ApoAl, ApoB, ApoM, APS (adaptor protein with pleckstrin homology and src homology 2 domains, a-synuclein, Aurora A, Aurora B, beta-1 integrin subunit, beta-amyloid converting enzyme (BACE), Bax, beta-catenin, Bc12, Bc1-XL, Bcr-abl, caspase 8, caspase-3, C CR2, CD40, CD4OL, cdk2, chk1, chk2, clottingfactorVII, collagen, CD132, CTLA4, cyclin E, Dhcr24, Dipeptidylpeptidase-IV, E-Cadherin, Eg5/KSP, EGF, EGFR1, EWS-Fli1, FAS-fatty acid synthase, FoxA-3, FoxO-1, Fructose-1,6-bisphosphate, Glucose-6-phophate, GM3 synthase, HDAC (histone deacetylase 1-6,9), Her-2/erb2, HIF1, HMG CoA reductase, hormone sensitive lipase, huntingtin, IKK1, IKK2, LDLR, MDR1, Microsomal Triglyceride Transfer Protein, MMPI, MMP2, MMP9, MyD88, sodium voltage gated type X alpha polypeptide (NaV1. 8), NFkB, p38 map kinase mitogen activated protein kinase, p85a regulatory subunit of PI3-kinase, PEPCK, plk1, PTEN, PTP-1B, PU. 1, raf, ras, Resistin, SCAP, SERBP-2, SHIP-2, SMAD7, SREBP1C, STAT1, stearoyl-CoA desaturase-1, TERT, TGF-beta-1, TGF-beta-1R1, Topoisomerase I, Topoisomerase II, VEGF, VEGFR1, VEGFR2, VLA1, VLA4, and vanilloid receptor (VR1).