Lipid encapsulated interfering RNA

Abstract

The present invention provides compositions and methods for silencing gene expression by delivering nucleic acid-lipid particles comprising a siRNA molecule to a cell.

1 . An nucleic acid-lipid particle, said nucleic acid-lipid particle comprising: 
 a siRNA;    a cationic lipid;    a non-cationic lipid; and    a conjugated lipid that inhibits aggregation of particles.    
     
     
         2 . The nucleic acid-lipid particle of  claim 1 , wherein said siRNA component of said nucleic acid-lipid particle is resistant in aqueous solution to degradation by a nuclease.  
     
     
         3 . The nucleic acid-lipid particle of  claim 1 , wherein said particle has a median diameter of less than about 150 nm.  
     
     
         4 . The nucleic acid-lipid particle of  claim 1 , wherein said siRNA comprises about 15 to about 60 nucleotides.  
     
     
         5 . The nucleic acid-lipid particle of  claim 1 , wherein said siRNA is transcribed from a plasmid encapsulated in said particle.  
     
     
         6 . The nucleic acid-lipid particle in accordance with  claim 1 , wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and combinations thereof.  
     
     
         7 . The nucleic acid-lipid particle in accordance with  claim 1 , wherein said non-cationic lipid is a member selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and combinations thereof.  
     
     
         8 . The nucleic acid-lipid particle in accordance with  claim 1 , wherein the conjugated lipid that inhibits aggregation of particles is a member selected from the group consisting of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and combinations thereof.  
     
     
         9 . The nucleic acid-lipid particle in accordance with  claim 1 , wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol-lipid and the PEG-lipid is member selected from the group consisting of a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and combinations thereof.  
     
     
         10 . The nucleic acid-lipid particle in accordance with  claim 9 , wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.  
     
     
         11 . The nucleic acid-lipid particle in accordance with  claim 10 , wherein the PEG-dialkyloxypropyl conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C 12 ), a PEG-dimyristyloxypropyl (C 14 ), a PEG-dipalmityloxypropyl (C 16 ), and a PEG-distearyloxypropyl (C 18 ), and combinations thereof.  
     
     
         12 . A method of introducing an siRNA into a cell, said method comprising contacting said cell with a nucleic acid-lipid particle comprising 
 a siRNA;    a cationic lipid;    a non-cationic lipid; and    a conjugated lipid that inhibits aggregation of particles.    
     
     
         13 . The method of  claim 12 , wherein said siRNA component of said nucleic acid-lipid particle is resistant in aqueous solution to degradation with a nuclease.  
     
     
         14 . The method of  claim 12 , wherein said particle has a median diameter of less than about 150 nm.  
     
     
         15 . The method of  claim 12 , wherein said siRNA is transcribed from a plasmid encapsulated in said particle.  
     
     
         16 . The method of  claim 12 , wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and combinations thereof.  
     
     
         17 . The method of  claim 12 , wherein said non-cationic lipid is a member selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and combinations thereof.  
     
     
         18 . The method of  claim 12 , wherein the conjugated lipid that inhibits aggregation of particles is a member selected from the group consisting of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and combinations thereof.  
     
     
         19 . The method of  claim 12 , wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-lipid and the PEG-lipid is member selected from the group consisting of a PEG-diacylglycerol, a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and combinations thereof.  
     
     
         20 . The method of  claim 12 , wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.  
     
     
         21 . The method of  claim 20 , wherein the PEG-dialkyloxypropyl conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C 12 ), a PEG-dimyristyloxypropyl (C 14 ), a PEG-dipalmityloxypropyl (C 16 ), and a PEG-distearyloxypropyl (C 18 ).  
     
     
         22 . The method of  claim 12 , wherein the presence of said nucleic acid-lipid particle in target cells is detectable at least 48 hours after administration of said particle.  
     
     
         23 . The method of  claim 12 , wherein the presence of said nucleic acid-lipid particle is detectable at least 24 hours after administration of said particle.  
     
     
         24 . The method of  claim 12 , wherein said cell is in a mammal.  
     
     
         25 . The method of  claim 24 , wherein more than 10% of such particles are present in plasma of said mammal 24 hours after administration.  
     
     
         26 . The method of  claim 24 , wherein presence of a siRNA at a site distal to the site of administration is detectable for at least 48 hours after administration of said particle.  
     
     
         27 . The method of  claim 24 , wherein presence of a siRNA at a site distal to the site of administration is detectable for at least 24 hours after administration of said particle.  
     
     
         28 . The method of  claim 27 , wherein the mammal is a human.  
     
     
         29 . A method for silencing expression of a target sequence, said method comprising administering to a mammalian subject an effect amount of a a therapeutically effective amount of a nucleic acid-lipid particle comprising: 
 a siRNA;    a cationic lipid;    a non-cationic lipid; and    a conjugated lipid that inhibits aggregation of particles.    
     
     
         30 . The method of  claim 29 , wherein said administration is intravenous.  
     
     
         31 . The method of  claim 29 , wherein said mammal is a human.  
     
     
         32 . The method of  claim 31 , wherein said human has a disease or disorder associated with expression of a gene comprising a target sequence for said siRNA.  
     
     
         33 . The method of  claim 32 , wherein said disease or disorder is hepatitis.  
     
     
         34 . A method for in vivo delivery of siRNA, said method comprising administering to a mammalian subject a nucleic acid-lipid particle comprising: 
 a siRNA;    a cationic lipid;    a non-cationic lipid; and    a conjugated lipid that inhibits aggregation of particles.    
     
     
         35 . The method of  claim 34 , wherein said administration is intravenous.  
     
     
         36 . The method of  claim 34 , wherein said mammal is a human.  
     
     
         37 . The method of  claim 36 , wherein said human has a disease or disorder associated with expression of a gene comprising a target sequence for said siRNA.  
     
     
         38 . The method of  claim 37 , wherein said disease or disorder is associated with overexpression of said gene.  
     
     
         39 . The method of  claim 37 , wherein said disease or disorder is hepatitis.  
     
     
         40 . A method for in vivo delivery of siRNA to a liver cell, said method comprising administering to a mammalian, subject a nucleic acid-lipid particle comprising: 
 a siRNA;    a cationic lipid;    a non-cationic lipid; and    a conjugated lipid that inhibits aggregation of particles.    
     
     
         41 . The method of  claim 40 , wherein said mammal is a human.  
     
     
         42 . The method of  claim 41 , wherein said human has a disease or disorder associated with expression of a gene comprising a target sequence for said siRNA.  
     
     
         43 . The method of  claim 42 , wherein said disease or disorder is associated with overexpression of said gene.  
     
     
         44 . The method of  claim 42 , wherein said disease or disorder is hepatitis.  
     
     
         45 . The method of  claim 40 , wherein said administration is intravenous.  
     
     
         46 . A method of treating a disease in a mammalian subject, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid-lipid particle comprising: 
 a siRNA;    a cationic lipid;    a non-cationic lipid; and    a conjugated lipid that inhibits aggregation of particles,    wherein said disease is associated with expression of a gene comprising a target sequence for said siRNA.    
     
     
         47 . The method of  claim 46 , wherein said mammal is a human.  
     
     
         48 . The method of  claim 46 , wherein said disease is hepatitis.  
     
     
         49 . The method of  claim 48 , wherein said hepatitis is Hepatitis B.  
     
     
         50 . The method of  claim 48 , wherein said hepatitis is Hepatitis C.  
     
     
         51 . The method of  claim 46 , wherein said administration is intravenous.