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.

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         97 . (A double-stranded ribonucleic acid (dsRNA) molecule comprising a sense strand and an anti-sense strand, and a cholesterol moiety linked to the end of one strand.  
     
     
         98 . The dsRNA molecule of  claim 1  wherein the dsRNA has a length of 15 to 40 base pairs.  
     
     
         99 . The dsRNA molecule of  claim 1  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 -3 aminopropylspermidine)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, N4 spermidine 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.  
     
     
         100 . The dsRNA of  claim 2 , wherein the cholesterol moiety is linked to the 3′ end of the antisense strand.  
     
     
         101 . The dsRNA molecule of  claim 4 , further comprising a second cholesterol moiety linked to the 3′ end of the sense strand.  
     
     
         102 . The dsRNA molecule of  claim 4 , wherein the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop.  
     
     
         103 . The dsRNA of  claim 2 , wherein the cholesterol moiety is linked to the 5′ end of the sense strand.  
     
     
         104 . The dsRNA molecule of  claim 7 , further comprising a second cholesterol moiety linked to the 5′ end of the antisense strand.  
     
     
         105 . The dsRNA molecule of  claim 7 , wherein the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop.  
     
     
         106 . A method for producing a double-stranded ribonucleic acid (dsRNA) molecule comprising hybridizing an sense and an antisense RNA strand together, each strand having a 5′ and a 3′ end, wherein a cholesterol moiety is linked to the end of one strand.  
     
     
         107 . The method for producing a dsRNA molecule of  claim 10 , wherein the dsRNA has a length of 15 to 40 base pairs.  
     
     
         108 . The method for producing a dsRNA molecule of  claim 10 , 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, N4 spermidine 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 method for producing a dsRNA molecule of  claim 10 , wherein the cholesterol moiety is linked to the 3′ end of the antisense strand.  
     
     
         110 . The method for producing a dsRNA molecule of  claim 13 , further comprising a second cholesterol moiety linked to the 3′ end of the sense strand.  
     
     
         111 . The method for producing a dsRNA molecule of  claim 13 , wherein the 3′ end of the sense strand is connected to the 5′ end of the antisense-strand by means of a loop.  
     
     
         112 . The method for producing a dsRNA molecule of  claim 10 , wherein the cholesterol moiety is linked to the 5′ end of the sense strand.  
     
     
         113 . The method for producing a dsRNA molecule of  claim 16 , further comprising a second cholesterol moiety linked to the 5′ end of the antisense strand.  
     
     
         114 . The method for producing a dsRNA molecule of  claim 16 , wherein the 3′ end of the sense strand is connected to the 5′ end of the antisense strand by means of a loop.  
     
     
         115 . A pharmaceutical composition for enhancing delivery of a double-stranded ribonucleic acid (dsRNA) into a cytoplasm of a mammalian target cell comprising: a dsRNA having a sense and an anti-sense strand, each strand having a 5′ and a 3′ end, a cholesterol moiety linked to the end of one strand; and a delivery-enhancing agent, wherein the enhancing agent is comprises at least one ingredient selected from the group consisting of: 
 (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;