Rna-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor

Abstract

The present invention relates to a novel, RNAi-inducing nucleic acid molecule having cell penetrating ability and the use thereof, and more particularly, to a novel, RNAi-inducing double-stranded nucleic acid molecule, which has a replacement of the phosphate backbone of at least one nucleotide with phosphorothioate or phosphorodithioate, and has a lipophilic compound conjugated thereto, and thus has high target gene-silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle, and to a method of silencing a target gene using the nucleic acid molecule. The nucleic acid structure according to the present invention has both cholesterol modification and phosphorothioate modification introduced therein, and thus has high gene silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle. Thus, it can be delivered into an actual target area in an amount sufficient for induction of RNAi, and thus can overcome the in vivo delivery problem occurring in the prior art. Therefore, the nucleic acid molecule according to the invention can effectively substitute for conventional siRNA molecules to treat cancer or viral infections.

1 . An RNAi-inducing double-stranded nucleic acid molecule having cell-penetrating ability, wherein the nucleic acid molecule comprising a first strand comprising a region complementary to a target nucleic acid and a second strand that forms a complementary bond with the first strand; and wherein the phosphate backbone of at least one nucleotide in the nucleic acid molecule was substituted with phosphorothioate or phosphorodithioate, and the nucleic acid molecule has a lipophilic compound conjugated thereto. 
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the RNAi-inducing double-stranded nucleic acid molecule comprises a first strand comprising a region complementary to a target nucleic acid, which is 24-121 nt in length, and a second strand which is 13-21 nt in length and comprises a region that binds complementarily to the region of the first strand, which is complementary to the target nucleic acid. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein the 5′ end of the first strand is a blunt end. 
     
     
         4 . The nucleic acid molecule of  claim 2 , wherein the region of the first strand, which is complementary to the target nucleic acid, is 19-31 nt in length. 
     
     
         5 . The nucleic acid molecule of  claim 1 , wherein the phosphate backbone(s) of 1 to 48 nucleotides is (are) substituted with phosphorothioate or phosphorodithioate. 
     
     
         6 . The nucleic acid molecule of  claim 5 , wherein the phosphate backbone(s) of 1 to 31 nucleotides in the first strand of the nucleic acid molecule is (are) substituted with phosphorothioate or phosphorodithioate. 
     
     
         7 . The nucleic acid molecule of  claim 5 , wherein the phosphate backbone(s) of 1 to 17 nucleotides in the second strand of the nucleic acid molecule is (are) substituted with phosphorothioate or phosphorodithioate. 
     
     
         8 . The nucleic acid molecule of  claim 7 , wherein the phosphate backbone(s) of nucleotides in a region of the first strand, other than a region complementary to a target nucleic acid, is (are) substituted with phosphorothioate or phosphorodithioate. 
     
     
         9 . The nucleic acid molecule of  claim 1 , wherein the phosphate backbone of at least one nucleotide in the nucleic acid molecule is substituted with phosphorothioate. 
     
     
         10 . The nucleic acid molecule of  claim 1 , wherein the lipophilic compound is selected from the group consisting of a lipid, a lipophilic peptide, and a lipophilic protein. 
     
     
         11 . The nucleic acid molecule of  claim 10 , wherein the lipid is at least one selected from the group consisting of cholesterol, tocopherol, and a long-chain fatty acid having 10 or more carbon atoms. 
     
     
         12 . The nucleic acid molecule of  claim 1 , wherein the lipophilic compound is conjugated to the end of the first or second strand of the nucleic acid molecule. 
     
     
         13 . The nucleic acid molecule of  claim 1 , wherein the target nucleic acid is at least one inducing RNAi selected from the group consisting of mRNA (messenger RNA), microRNA, piRNA (piwi-interacting RNA), a coding DNA sequence and a non-coding DNA sequence. 
     
     
         14 . The nucleic acid molecule of  claim 1 , hydroxyl group at position 2′ of ribose of at least one nucleotide included in the nucleic acid molecule is substituted with at least one selected from the group consisting of a hydrogen atom, a fluorine atom, an —O-alkyl group, an —O-acyl group and an amino group. 
     
     
         15 . The nucleic acid molecule of  claim 1 , wherein the phosphate backbone of at least one nucleotide, included in the nucleic acid molecule, is substituted with at least one selected from the group consisting of alkylphosphonate form, phosphoroamidate form and boranophosphate form. 
     
     
         16 . The nucleic acid molecule of  claim 1 , at least one nucleotide included in the nucleic acid molecule is substituted with at least one selected from the group consisting of LNA (locked nucleic acid), UNA (unlocked nucleic acid), morpholino and PNA (peptide nucleic acid). 
     
     
         17 . The nucleic acid molecule of  claim 1 , at least one of the nucleotides of the single-stranded region in the first strand comprises a bulky base analog. 
     
     
         18 . The nucleic acid molecule of  claim 1 , wherein the target nucleic acid is mRNA encoding a connective tissue growth factor (CTGF), and the RNAi-inducing double-stranded nucleic acid molecule has a pair of nucleic sequences selected from the group consisting of a pair of nucleotide sequences of SEQ ID NOS: 149 and 150, a pair of nucleotide sequences of SEQ ID NOS: 151 and 152, and a pair of nucleotide sequences of SEQ ID NOS: 153 and 154. 
     
     
         19 - 24 . (canceled)