In vivo delivery of oligonucleotides

Abstract

This invention provides a method for the in vivo delivery of oligonucleotides. The invention utilizes the presence of one or plurality of HES linked to an oligonucleotide to deliver a nucleic acid sequence of interest into the cytoplasm of cells and tissues of live organisms. The delivery vehicle is nontoxic to cells and organisms. Since delivery is sequence-independent and crosses membranes in a receptor-independent manner, the delivered oligonucleotide can target complementary sequences in the cytoplasm as well as in the nucleus of live cells. Sequences of bacterial or viral origin can also be targeted. The method can be used for delivery of genes coding for expression of specific proteins, antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, miRNAs, anti-miRNAs or any nucleic acid sequence in a living organism. The latter include mammals, plants, and microorganisms such as bacteria, protozoa, and viruses.

1 . A composition for delivering a therapeutic oligonucleotide to a subject, said composition comprising a therapeutically effective amount of an H-type excitonic structure (HES)-oligonucleotide containing a therapeutic oligonucleotide that specifically hybridizes to a nucleic acid sequence in vivo and modulates the level of a protein encoded or regulated by the nucleic acid. 
     
     
         2 . The composition of  claim 1 , wherein the therapeutic oligonucleotide is from about 8 nucleotides to about 750 nucleotides. 
     
     
         3 . The composition of  claim 1 , wherein the therapeutic oligonucleotide is single stranded or double stranded. 
     
     
         4 . (canceled) 
     
     
         5 . The composition of  claim 1 , wherein the HES-oligonucleotide comprises 3 or more fluorophores capable of forming one or more HES. 
     
     
         6 . The composition of  claim 1 , wherein the therapeutic oligonucleotide is a member selected from: siRNA, shRNA, miRNA, a Dicer substrate, an aptamer, a decoy and an antisense. 
     
     
         7 . The composition of  claim 6  wherein the therapeutic oligonucleotide is an antisense oligonucleotide that specifically hybridizes to an RNA. 
     
     
         8 . The composition of  claim 7 , wherein the therapeutic antisense oligonucleotide:
 (a) is a substrate for RNAse H when hybridized to the RNA,   (b) is not a substrate for RNAse H when hybridized to the RNA;   (c) is DNA or a DNA mimic; or   (d) is specifically hybridizable to a target region of the RNA selected from the group consisting of
 (i) a sequence within 30 nucleotides of the AUG start codon of an mRNA; 
 (ii) nucleotides 1-10 of a miRNA; 
 (iii) a sequence in the 5′ untranslated region of an mRNA; 
 (iv) a sequence in the 3′ untranslated region of an mRNA; 
 (v) an intron/exon junction of an mRNA; 
 (vi) a sequence in a precursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when bound by the oligonucleotide blocks miRNA processing; and 
 (vii) an intron/exon junction and a region 1 to 50 nucleobases 5′ of an intron/exon junction of an RNA. 
   
     
     
         9 .- 13 . (canceled) 
     
     
         14 . The composition of  claim 1 , wherein the therapeutic oligonucleotide can induce RNA interference (RNAi). 
     
     
         15 .- 16 . (canceled) 
     
     
         17 . The composition of claim  15 , wherein the therapeutic oligonucleotide is a dicer substrate and wherein the composition contains 2 complementary nucleic acid strands that are each 18-25 nucleotides in length and contain a 2 nucleotide 3′ overhang. 
     
     
         18 . The composition according to  claim 1  that contains one or more modified nucleoside motifs selected from: 2′OME, locked nucleic acid (LNA), alpha LNA, 2-Fluoro (2′F), 2′-O(CH 2 ) 2 OCH 3  (2′-MOE), 2′-OCH 3  (2′-O-methyl), PNA and morpholino. 
     
     
         19 .- 20 . (canceled) 
     
     
         21 . The composition according to  claim 1  that contains one or more selected from: a phosphorothioate, a phosphorodithioate, a phosphoramide, a 3′-methylene phosphonate, a O-methylphosphoroamidiate, a PNA, a morpholino, a C-5 propyne and a 5-methyl C. 
     
     
         22 .- 24 . (canceled) 
     
     
         25 . A method for modulating a target nucleic acid or protein in a subject, said method comprising administering to the subject a therapeutically effective amount of an H-type excitonic structure (HES)-oligonucleotide containing a therapeutic oligonucleotide that specifically hybridizes to a target nucleic acid sequence in vivo and thereby modulates the level of a protein encoded or regulated by the nucleic acid. 
     
     
         26 . The method of  claim 25  wherein the disease or disorder is selected from the group consisting of: an infectious disease, cancer, a proliferative disease or disorder, a neurological disease or disorder, and inflammatory disease or disorder, a disease or disorder of the immune system, a disease or disorder of the cardiovascular system, a metabolic disease or disorder, a disease or disorder of the skeletal system, and a disease or disorder of the skin or eyes. 
     
     
         27 . A method for modulating a target nucleic acid or protein in a cell ex vivo, said method comprising administering to the cell a therapeutically effective amount of an H-type excitonic structure (HES)-oligonucleotide containing a therapeutic oligonucleotide that specifically hybridizes to the target nucleic acid sequence in the cell and thereby modulates the level of protein encoded or regulated by the nucleic acid. 
     
     
         28 . (canceled) 
     
     
         29 . A method for treating a disease or disorder in a subject, said method comprising administering to a subject in need thereof, a therapeutically effective amount of the composition of  claim 1 ;
 wherein the disease or disorder is characterized by   (a) overexpression or underexpression of the target a nucleic acid in the subject, or   (b) overexpression or underexpression of a protein encoded by the target nucleic in the subject.   
     
     
         30 . The composition of  claim 8 , wherein the therapeutic oligonucleotide is siRNA, shRNA or a Dicer substrate. 
     
     
         31 . The composition of  claim 8 , wherein the therapeutic oligonucleotide is a Dicer substrate 18-35 nucleotides in length.