Cell transfecting formulations of small interfering rna, related compositions and methods of making and use

Abstract

Compositions incorporating small interfering ribonucleic acid (siRNA) and certain lipid-conjugated polyamide compound-based delivery vehicles that are particularly useful in the delivery siRNA and other polynucleotides to cells. Also, methods of making and using the compositions.

What is claimed is: 
     
         1 . A composition, comprising:
 a small interfering ribonucleic acid (siRNA) in a pharmaceutically acceptable delivery vehicle, the vehicle comprising at least one compound of the formula:
   R a —[(NR 1 —W—CO) n ] m —R c  
 
   wherein n is an integer selected from 1 to about 48 and m is an integer selected from about 2 to about 48,   wherein R 1  for each monomeric unit, —(NR 1 —W—CO)—, and R a  are independently selected from the group consisting of a hydrogen atom; a hydroxy group; an amino group; a carboxyl group; a sulfonyl group; —SH; an optionally substituted, branched or straight chain aliphatic group having 1 to 8 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein said aliphatic group optionally has one or more double or triple bonds; an optionally substituted aryl group having 3 to 12 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus; an optionally substituted arylalkyl group having 3 to 12 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein the alkyl group of said arylalkyl optionally has one or more double or triple bonds; and a lipid moiety that is optionally bonded to linker moiety,   wherein R 1  is not a hydrogen atom for at least one monomeric unit,   wherein R c  is selected from a hydrogen atom; a hydroxy group; an amino group; a hydrazine group; a sulfonyl group; —SH; an optionally substituted, branched or straight chain aliphatic group having 1 to 8 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein said aliphatic group optionally has one or more double or triple bonds; an optionally substituted aryl group having 3 to 12 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus; an optionally substituted arylalkyl group having 3 to 12 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein the alkyl group of said arylalkyl optionally has one or more double or triple bonds; and a lipid moiety that is optionally bonded to a linker moiety,   wherein when R 1 , R a , or R c  is an aryl or arylalkyl group having fewer than 5 carbon atoms in a backbone structure, said backbone structure further comprises one or more oxygen and/or nitrogen atoms,   wherein W for each monomeric unit is independently selected from an optionally substituted, branched or straight chain divalent moiety having from 1 to about 50 atoms and optionally, one or more double or triple bonds in a backbone that contains carbon and optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein said optional substitution of W may be a lipid moiety that is optionally bonded to a linker moiety,   wherein said lipid moiety is a hydrophobic or amphipathic moiety selected from the group consisting of:   (i) optionally substituted aryl or arylalkyl moieties having from about 14 to about 50 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein the alkyl group of said arylalkyl optionally has one or more double or triple bonds; and   (ii) optionally substituted, branched or straight chain aliphatic moieties having from about 10 to about 50 carbon atoms in a backbone structure that optionally contains nitrogen, oxygen, sulfur, and phosphorus, wherein said aliphatic moieties optionally have one or more double or triple bonds, and   wherein at least one of R a , R c , W for a single monomeric unit and R 1  for a single monomeric unit comprises a lipid moiety.   
     
     
         2 . The composition of  claim 1 , wherein the delivery vehicle comprises a lipid-cationic peptoid conjugate of the formula:
   L-X—[N(CH 2 CH 2 NH 2 )CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)] 3 —NH 2  
   
       and positional isomers 
       where
 L is selected from a non-sterol lipid moiety comprising at least one fatty alkyl or alkenyl chain between about 8 and 24 carbon atoms in length and a sterol moiety; 
 each group R is independently selected from alkyl, aminoalkyl, and aralkyl, and 
 X is selected from the group consisting of a direct bond, an oligopeptide, a substantially linear alkyl chain from 2 to about 30 bonds in length, and a substantially linear chain from 2 to about 30 bonds in length consisting of alkyl bonds and one or more linkages selected from the group consisting of ester, amide, carbonate, carbamate, disulfide, peptide, and ether. 
 
     
     
         3 . The composition of  claim 1 , wherein said fatty alkyl or alkenyl chain is between about 14 and 24 carbon atoms in length. 
     
     
         4 . The composition of  claim 1 , wherein L is a phospholipid group, having two fatty alkyl or alkenyl chains between about 8 and 24 carbon atoms in length. 
     
     
         5 . The composition of  claim 1 , wherein L is a cholesteryl group. 
     
     
         6 . The composition of  claim 1 , wherein R is isopropyl or 4-methoxyphenyl. 
     
     
         7 . The composition of  claim 1 , wherein the delivery vehicle comprises a lipid-cationic peptoid conjugate is of the formula:
   L-(CH 2 ) n —(C═O)—[N(CH 2 CH 2 NH 2 )CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)] 3 —NH 2  
   
       where
 L is selected from (i) a phosphatidylethanolamino group, having fatty alkyl or alkenyl chains between about 8 and 24 carbon atoms in length, and (ii) a cholesteryl group linked to the adjacent —(CH 2 ) n — segment by an ester, amide or carbamate linkage; 
 n is 1-5; and 
 R is selected from isopropyl and 4-methoxyphenyl. 
 
     
     
         8 . The composition of  claim 2 , wherein the lipid-cationic peptoid conjugate is selected from the group consisting of compounds represented herein as: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Lipitoid 1, or L1 
                   DMPE(NaeNmpeNmpe) 3   
                 
                     
                   Lipitoid 2, or L2 
                   DMPE(NaeNiaNia) 3   
                 
                     
                   Lipitoid 3, or L3 
                   NtdNhd(NaeNmpeNmpe) 3   
                 
                     
                   Lipitoid 4, or L4 
                   NddNol(NaeNmpeNmpe) 3   
                 
                     
                   Cholesteroid 1, or C1 
                   Chol-β-ala-(NaeNmpeNmpe) 3   
                 
                     
                   Cholesteroid 3, or C3 
                   Chol-β-ala-(NaeNiaNia) 3   
                 
                     
                     
                 
             
                
               
               
                
                
                
                
                
                
                
               
            
           
         
       
       and combinations thereof. 
     
     
         9 . The composition of  claim 8 , wherein the lipid-cationic peptoid conjugate is a combination of a lipitoid and a cholesteroid. 
     
     
         10 . The composition of  claim 9 , wherein the lipitoid:cholesteroid ratio is about 5:1 to 1:5. 
     
     
         11 . The composition of  claim 10 , wherein the lipitoid:cholesteroid ratio is about 1:1 to 1:3. 
     
     
         12 . The composition of  claim 11 , wherein the lipitoid is L1 and the cholesteroid is C1. 
     
     
         13 . The composition of  claim 11 , wherein the lipitoid:cholesteroid ratio is about 1:3. 
     
     
         14 . The composition of  claim 1 , wherein the vehicle:siRNA molar ratio is about 3.75 nmol:100 pmol. 
     
     
         15 . The composition of  claim 1 , wherein the siRNA comprises a 21-23 mer RNA duplex with two nucleotide 3′ overhangs and phophodiester links throughout. 
     
     
         16 . The composition of  claim 15 , wherein the siRNA comprises the sequence represented by SEQ ID NO: 1, with two nucleotide 3′-overhangs and phosphodiester links throughout. 
     
     
         17 . A composition, comprising:
 a mixture of at least two lipid-cationic peptoid conjugates, each of the formula:
   L-X—[N(CH 2 CH 2 NH 2 )CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)] 3 —NH 2  
 
   and positional isomers,   wherein, in a first of the compounds, L is a non-sterol lipid moiety comprising at least one fatty alkyl or alkenyl chain between about 8 and 24 carbon atoms in length, and, in a second of the compounds, L is a sterol moiety;   each group R is independently selected from alkyl, aminoalkyl, and aralkyl, and   X is selected from the group consisting of a direct bond, an oligopeptide, a substantially linear alkyl chain from 2 to about 30 bonds in length, and a substantially linear chain from 2 to about 30 bonds in length consisting of alkyl bonds and one or more linkages selected from the group consisting of ester, amide, carbonate, carbamate, disulfide, peptide, and ether.   
     
     
         18 . The composition of  claim 17 , wherein the ratio of the first to the second compounds in the composition is from about 5:1 to 1:5. 
     
     
         19 . The composition of  claim 18 , wherein the ratio of the first to the second compounds in the composition is from about 1:1 to 1:3. 
     
     
         20 . The composition of  claim 17 , wherein the ratio of the first to the second compounds in the composition is about 1:1. 
     
     
         21 . The composition of  claim 17 , wherein the ratio of the first to the second compounds in the composition is from about 1:3. 
     
     
         22 . The composition of  claim 17 , wherein the lipid-cationic peptoid conjugates are independently selected from the group consisting of compounds represented herein as: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Lipitoid 1, or L1 
                   DMPE(NaeNmpeNmpe) 3   
                 
                     
                   Lipitoid 2, or L2 
                   DMPE(NaeNiaNia) 3   
                 
                     
                   Lipitoid 3, or L3 
                   NtdNhd(NaeNmpeNmpe) 3   
                 
                     
                   Lipitoid 4, or L4 
                   NddNol(NaeNmpeNmpe) 3   
                 
                     
                   Cholesteroid 1, or C1 
                   Chol-β-ala-(NaeNmpeNmpe) 3   
                 
                     
                   Cholesteroid 3, or C3 
                   Chol-β-ala-(NaeNiaNia) 3   
                 
                     
                     
                 
             
                
               
               
                
                
                
                
                
                
                
               
            
           
         
       
       and combinations thereof. 
     
     
         23 . The composition of  claim 22 , wherein the composition consists of L1 and C1.