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

Cholesterol moieties are linked to specific ends of double-stranded RNA, preferably a small, interfering (si)RNA or to a dsHybrid. The dsHybrid has one strand comprised of DNA and one strand comprised of RNA. Preferably the sense strand is the DNA strand and the antisense strand is the RNA strand of the dsHybrid. The present invention is based upon the discovery that a cholesterol moiety, if linked to a specific end or ends of the sense or antisense strands of a siRNA, can enhance the delivery and silencing efficiency of the siRNA directed against its target message, in comparison with a corresponding, non-conjugated siRNA. Conjugated siRNAs and dsHybrids of the invention are optionally formulated with, or coordinately administered with, a secondary delivery-enhancing agent, such as a delivery-enhancing peptide, to enhance intracellular delivery and uptake of the conjugated siRNAs or dsHybrid.

1 .- 96 . (canceled) 
     
     
         97 . A double-stranded (ds) RNA or a dsHybrid having a sense and an anti-sense strand, each strand having a 5′ and a 3′ end, wherein:
 (a) a cholesterol moiety is linked to the 5′ end of the sense strand and a cholesterol moiety is linked to the 5′ end of the antisense and wherein no cholesterol moiety is linked to the other ends of the strands; 
 (b) a cholesterol moiety is linked to the 3′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands; 
 (c) a cholesterol moiety is linked to the 5′ end of the sense strand and no cholesterol moiety is linked to the other ends of the strands; 
 (d) a cholesterol moiety is linked to the 3′ end of the sense strand and no cholesterol moiety is linked to the other ends of the strands; 
 (e) a cholesterol moiety is linked to the 3′ end of the sense strand, a cholesterol moiety is linked to the 3′ end of the antisense strand and no cholesterol moiety is linked to the other ends of the strands; 
 a cholesterol moiety is linked to the 5′ end of the antisense strand and no cholesterol moiety is linked to the other ends of the strands; 
 (g) the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop, and wherein a cholesterol moiety is linked to the 3′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands; and/or 
 (h) the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop, wherein a cholesterol moiety is linked to the 5′ end of the sense strand and no cholesterol moiety is linked to the other ends of the strands. 
 
     
     
         98 . The dsRNA or dsHybrid of  claim 97  wherein the dsRNA or dsHybrid has a length of 19 to 21 base pairs. 
     
     
         99 . The dsHybrid of  claim 97  wherein the sense strand is a DNA strand. 
     
     
         100 . The dsRNA or dsHybrid of  claim 97  wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, any 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 and lithocholic acid and derivative thereof. 
     
     
         101 . A method for producing double-stranded (ds) RNA comprising hybridizing an RNA sense and an RNA antisense strand together, each strand having a 5′ and a 3′end, wherein:
 (a) a cholesterol moiety is linked to the 5′ end of the sense strand and a cholesterol moiety is linked to the 5′ end of the antisense and wherein no cholesterol moiety is linked to the other ends of the strands; 
 (b) a cholesterol moiety is linked to the 3′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands; 
 (c) a cholesterol moiety is linked to the 5′ end of the sense strand and no cholesterol moiety is linked to the other ends of the siRNA strands; 
 (d) a cholesterol moiety is linked to the 3′ end of the sense strand and no cholesterol moiety is linked to the other ends of the siRNA strands; 
 (e) a cholesterol moiety is linked to the 3′ end of the sense strand, a cholesterol moiety is linked to the 3′ end of the antisense strand and no cholesterol moiety linked to the other ends of the siRNA or strands; 
 (f) a cholesterol moiety is linked to the 5′ end of the antisense strand and no cholesterol moiety is linked to the other ends of the dsRNA strands; 
 (g) the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop, and wherein a cholesterol moiety is linked to the 3′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands of the dsRNA a cholesterol moiety is linked to the 5′ end of the sense strand and no cholesterol moiety is linked to the other ends of the dsRNA strands; 
 (h) a cholesterol moiety is linked to the 5′ end of the sense strand and a cholesterol moiety is linked to the 5′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands; 
 (i) a cholesterol moiety is linked to the 3′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands; 
 (j) a cholesterol moiety is linked to the 5′ end of the sense strand and no cholesterol moiety is linked to the other ends of the strands; 
 (k) a cholesterol moiety is linked to the 3′ end of the sense strand and no cholesterol moiety is linked to the other ends of the strands; 
 (l) a cholesterol moiety is linked to the 3′ end of the sense strand, a cholesterol moiety is linked to the 3′ end of the antisense strand and no cholesterol moiety is linked to the other ends of the strands; 
 (m) a cholesterol moiety is linked to the 5′ end of the antisense strand and no cholesterol moiety is linked to the other ends of the dsRNA strands; 
 (n) the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop, and wherein a cholesterol moiety is linked to the 3′ end of the antisense strand and wherein no cholesterol moiety is linked to the other ends of the strands of the dsHybrid; and/or 
 (o) the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop, wherein a cholesterol moiety is linked to the 5′ end of the sense strand and no cholesterol moiety linked to the other ends of the dsHybrid strands. 
 
     
     
         102 . The method of  claim 101  wherein the dsRNA has a length of 19 to 21 base pairs. 
     
     
         103 . The method of  claim 101  wherein the cholesterol moiety is selected from the group consisting of cholesterol, sterol, any 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 6 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 and lithocholic acid and derivative thereof. 
     
     
         104 . A composition for enhancing delivery of a double-stranded (ds) RNA or a dsHybrid into a cytoplasm of a mammalian target cell, said composition comprising:
 (a) a double-stranded (ds) RNA or dsHybrid and   (b) one or more secondary delivery-enhancing agent(s) selected from:
 (i) an aggregation inhibitory agent; 
 (ii) a charge modifying agent; 
 (iii) a pH control agent; 
 (iv) a degradative enzyme inhibitory agent; 
 (v) a mucolytic or mucus clearing agent; 
 (vi) a ciliostatic agent; 
 (vii) a membrane penetration-enhancing agent selected from a surfactant, a bile salt, a phospholipid additive, a mixed micelle, a liposome, a carrier an alcohol, an enamine, an NO donor compound, a long-chain amphipathic molecule, a small hydrophobic penetration enhancer, sodium or a salicylic acid derivative, a glycerol ester of acetoacetic acid, a cyclodextrin or beta-cyclodextrin derivative, a medium-chain fatty acid, a chelating agent, an amino acid or salt thereof, an N-acetylamino acid or salt thereof, an enzyme degradative to a selected membrane component, an inhibitor of fatty acid synthesis, and/or an inhibitor of cholesterol synthesis; 
 (viii) a delivery-enhancing peptide; 
 (ix) a vasodilator agent; 
 (x) a selective transport-enhancing agent; and 
 (xi) a stabilizing delivery vehicle, a carrier, a support, and/or a complex-forming species. 
   
     
     
         105 . The composition of  claim 104  wherein the dsRNA or dsHybrid has a length of 19 to 21 base pairs. 
     
     
         106 . The composition of  claim 104  wherein the sense strand is a DNA strand. 
     
     
         107 . The composition of  claim 104  wherein said double-stranded (ds) RNA or dsHybrid comprises a sense and an anti-sense strand, each strand having a 5′ and a 3′ end, wherein a cholesterol moiety is linked to the 5′ ends of the sense and antisense strands or to the 3′ ends of the sense and antisense strands. 
     
     
         108 . The composition of  claim 107  wherein said cholesterol moiety is selected from the group consisting of cholesterol, sterol, any 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 and lithocholic acid and derivative thereof. 
     
     
         109 . The composition of  claim 104  wherein the secondary delivery-enhancing agent(s) include(s) a delivery-enhancing peptide. 
     
     
         110 . The composition of  claim 104  wherein the delivery-enhancing peptide comprises an amino acid sequence selected from: 
       
         
           
                 
               
                   (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 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         111 . The composition of  claim 104  wherein the delivery-enhancing peptide is combined with or conjugated to the double-stranded (ds) RNA or dsHybrid linked to the cholesterol moiety. 
     
     
         112 . The composition of  claim 104  which is effective to deliver the double-stranded (ds) RNA or dsHybrid linked to the cholesterol moiety to a target mammalian cell selected from pulmonary alveolar cells, skin cells, hepatic cells, renal cells, pancreatic cells, endothelial cells, nucleated blood cells, muscle cells, mammary cells, peripheral or central nervous system (CNS) cells, gastrointestinal cells, or tumor cells.