Methods and compositions for enhancing delivery of double-stranded rna or a double-stranded hybrid nucleic acid to regulate gene expression in mammalian cells

Abstract

A double-stranded RNA, preferably a small, interfering (si)RNA or a siHybrid, to which cholesterol moieties are linked.

1 . A double-stranded RNA which is active for RNA interference having a sense and an anti-sense strand, each strand having a 5′ end and a 3′ end, wherein a cholesterol moiety is linked to the 5′ end of the antisense strand. 
     
     
         2 . The double-stranded RNA of  claim 1  having a length of 19 to 21 base pairs. 
     
     
         3 . The double-stranded RNA of  claim 1 , wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, a compound derived from cholesterol, chlolestanol, ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid, cortisol, corticosterone, Δ 5 -pregnenolone, progesterone, deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone, 9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol, estrone, estradiol, estrogen, spermidine cholesterol carbamate, N 4 -spermidine cholesteryl carbamate, N 4 -spermidine cholesteryl carbamate di HCl salt, N 4 -spermidine-7 dehydro cholesteryl carbamate, N 4 -spermine cholesteryl carbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,N bis(6-aminohexyl) cholesteryl carbamate, N 4 -spermidine dihydrocholesteryl carbamate, N 4 -spermidine lithocholic carbamate methyl ester, N 1 ,N 8 -bis(3-aminopropyl-N 4 -spermidine cholesteryl carbamate, N(N 4 -3aminopropylspermidine) cholesteryl carbamate, N,N-bis(4-aminobutyl) cholesteryl carbamate, N 4 -spermidine cholesteryl urea, N 4 -spermine cholesteryl urea, N 4 -spermidine dihydro cholesteryl urea, N 4 -spermine dihydro cholesteryl urea, N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate, N4spermidine cholesteryl carboxamide, and N—[N 1 ,N 4 ,N 8 -tris (3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid, and derivatives thereof. 
     
     
         4 . An siHybrid having a sense strand and an antisense strand, each strand having a 5′ end and a 3′ end, wherein a cholesterol moiety is linked to the 3′ end of the antisense strand. 
     
     
         5 . The siHybrid of  claim 4  having a length of 19 to 21 base pairs. 
     
     
         6 . The siHybrid of  claim 4 , wherein the sense strand is a DNA strand. 
     
     
         7 . The siHybrid of  claim 4 , wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, a compound derived from cholesterol, chlolestanol, ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid, cortisol, corticosterone, Δ 5 -pregnenolone, progesterone, deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone, 9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol, estrone, estradiol, estrogen, spermidine cholesterol carbamate, N 4 -spermidine cholesteryl carbamate, N 4 -spermidine cholesteryl carbamate di HCl salt, N 4 -spermidine-7 dehydro cholesteryl carbamate, N 4 -spermine cholesteryl carbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,N bis(6-aminohexyl) cholesteryl carbamate, N 4 -spermidine dihydrocholesteryl carbamate, N 4 -spermidine lithocholic carbamate methyl ester, N 1 ,N 8 -bis(3-aminopropyl-N 4 -spermidine cholesteryl carbamate, N(N 4 -3aminopropylspermidine) cholesteryl carbamate, N,N-bis(4-aminobutyl) cholesteryl carbamate, N 4 -spermidine cholesteryl urea, N 4 -spermine cholesteryl urea, N 4 -spermidine dihydro cholesteryl urea, N 4 -spermine dihydro cholesteryl urea, N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate, N4spermidine cholesteryl carboxamide, and N—[N 1 ,N 4 ,N 8 -tris (3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid, and derivatives thereof. 
     
     
         8 . A composition for delivering a double-stranded RNA to a mammalian cell comprising a double-stranded RNA having a sense and an anti-sense strand, each strand having a 5′ end and a 3′ end, wherein a cholesterol moiety is linked to the 5′ end of the antisense strand, and one or more delivery-enhancing agents selected from:
 (a) an aggregation inhibitory agent;   (b) a charge modifying agent;   (c) a pH control agent;   (d) a degradative enzyme inhibitory agent;   (e) a mucolytic or mucus clearing agent;   (f) a ciliostatic agent;   (g) a membrane penetration-enhancing agent selected from (i) a surfactant, (ii) a bile salt, (iii) a phospholipid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chain amphipathic molecule (viii) a small hydrophobic penetration enhancer; (ix) sodium or a salicylic acid derivative; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xv) an N-acetylamino acid or salt thereof, (xvi) an enzyme degradative to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, or (xviii) an inhibitor of cholesterol synthesis; or (xix) any combination of the membrane penetration enhancing agents recited in (i)-(xviii);   (h) a delivery-enhancing peptide;   (i) a vasodilator agent;   (j) a selective transport-enhancing agent; and   (k) a RNA-stabilizing vehicle or carrier.   
     
     
         9 . The composition of  claim 8 , wherein the double-stranded RNA has a length of 19 to 21 base pairs. 
     
     
         10 . The composition of  claim 8 , wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, a compound derived from cholesterol, chlolestanol, ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid, cortisol, corticosterone, Δ 5 -pregnenolone, progesterone, deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone, 11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6α-methylpredisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylprednisolone, 9α-fluorocortisol, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α,5α-androstanediol, estrone, estradiol, estrogen, spermidine cholesterol carbamate, N 4 -spermidine cholesteryl carbamate, N 4 -spermidine cholesteryl carbamate di HCl salt, N 4 -spermidine-7 dehydro cholesteryl carbamate, N4-spermine cholesteryl carbamate, N,N bis(3-aminopropyl) cholesteryl carbamate, N,N bis(6-aminohexyl) cholesteryl carbamate, N 4 -spermidine dihydrocholesteryl carbamate, N 4 -spermidine lithocholic carbamate methyl ester, N 1 ,N 8 -bis(3-aminopropyl-N 4 -spermidine cholesteryl carbamate, N(N 4 -3aminopropylspermidine) cholesteryl carbamate, N,N-bis(4-aminobutyl) cholesteryl carbamate, N 4 -spermidine cholesteryl urea, N 4 -spermine cholesteryl urea, N 4 -spermidine dihydro cholesteryl urea, N 4 -spermine dihydro cholesteryl urea, N,N-bis(N′-3-aminopropyl-N″4-aminobutyl) cholesteryl carbamate, N4spermidine cholesteryl carboxamide, and N—[N 1 ,N 4 ,N 8 -tris (3-aminopropyl)spermidine] cholesteryl carbamate, lumisterol, cholic acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid, and derivatives thereof. 
     
     
         11 . The composition of  claim 8 , wherein the delivery-enhancing agent is a delivery-enhancing peptide having the amino acid sequence selected from the group of: 
       
         
           
                 
                 
               
                   (SEQ ID NO: 1) 
                     
                 
                 
                 
                 
               
                     
                   RKKRRQRRRPPQCAAVALLPAVLLALLAP; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 2) 
                     
                 
                 
                 
                 
               
                     
                   RQIKIWFQNRRMKWKK; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 3) 
                     
                 
                 
                 
                 
               
                     
                   GWTLNSAGYLLGKINLKALAALAKKIL; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 4) 
                     
                 
                 
                 
                 
               
                     
                   KLALKLALKALKAALKLA; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 7) 
                     
                 
                 
                 
                 
               
                     
                   KLWSAWPSLWSSLWKP; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 8) 
                     
                 
                 
                 
                 
               
                     
                   AAVALLPAVLLALLAPRKKRRQRRRPPQ; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 9) 
                     
                 
                 
                 
                 
               
                     
                   LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 10) 
                     
                 
                 
                 
                 
               
                     
                   RRRQRRKRGGDIMGEWGNEIFGAIAGFLG; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 11) 
                     
                 
                 
                 
                 
               
                     
                   KETWWETWWTEWSQPGRKKRRQRRRPPQ; 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 12) 
                     
                 
                 
                 
                 
               
                     
                   GLGSLLKKAGKKLKQPKSKRKV; 
                     
                 
                     
                   and 
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO: 13) 
                     
                 
                 
                 
                 
               
                     
                   KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ. 
                     
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         12 . The composition of  claim 11 , wherein the delivery-enhancing peptide is conjugated to the double-stranded RNA. 
     
     
         13 . The composition of  claim 8 , wherein the mammalian cell is selected from a pulmonary alveolar cell, a skin cell, an hepatic cell, a renal cell, a pancreatic cell, an endothelial cell, a nucleated blood cell, a muscle cell, a mammary cell, a peripheral cell, a central nervous system cell, a gastrointestinal cell, and a tumor cell.