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.

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 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 DTMPE(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 method of inhibiting expression of a target gene in a subject, comprising administering to the subject a composition as recited in  claim 1 , wherein one strand of the siRNA duplex has a nucleotide sequence comprised in a mRNA derived from the target gene.  
     
     
         18 . The method of  claim 17 , wherein the delivery vehicle comprises a lipid-cationic peptoid conjugate as recited in  claim 2 .  
     
     
         19 . The method of  claim 18 , wherein the delivery vehicle is a combination of a lipitoid and a cholesteroid.  
     
     
         20 . The method of  claim 19 , wherein the lipitoid:cholesteroid ratio is about 5:1 to 1:5.  
     
     
         21 . The method of  claim 20 , wherein the lipitoid:cholesteroid ratio is about 1:1 to 1:3.  
     
     
         22 . The method of  claim 21 , wherein the lipitoid is L1 and the cholesteroid is C1.  
     
     
         23 . The method of  claim 17 , wherein the siRNA comprises the sequence represented by SEQ ID NO: 1 with two nucleotide 3′-overhangs and phosphodiester links throughout.  
     
     
         24 . The method of  claim 17 , wherein the subject is a mammalian cell or cell line.  
     
     
         25 . The method of  claim 17 , wherein the subject is a mammal.  
     
     
         26 . The method of  claim 25 , wherein the subject is a human.  
     
     
         27 . 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.    
     
     
         28 . The composition of  claim 27 , wherein the ratio of the first to the second compounds in the composition is from about 5:1 to 1:5.  
     
     
         29 . The composition of  claim 28 , wherein the ratio of the first to the second compounds in the composition is from about 1:1 to 1:3.  
     
     
         30 . The composition of  claim 2 , wherein the ratio of the first to the second compounds in the composition is about 1:1.  
     
     
         31 . The composition of  claim 2 , wherein the ratio of the first to the second compounds in the composition is from about 1:3.  
     
     
         32 . The composition of  claim 27 , 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.    
     
     
         33 . The composition of  claim 32 , wherein the composition consists of L1 and C1.