Methods and compositions for RNAi mediated inhibition of gene expression in mammals

Abstract

Methods and compositions are provided for modulating, e.g., reducing, coding sequence expression in mammals. In the subject methods, an effective amount of an RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is administered to a non-embryonic mammal, e.g., via a hydrodynamic administration protocol. Also provided are RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including academic and therapeutic applications.

What is claimed is:  
     
         1 . A method of reducing expression of a coding sequence in a target cell of a non-embryonic mammal, said method comprising: 
 administering to said mammal an effective amount of an RNAi agent specific for said coding sequence to reduce expression of said coding sequence.    
     
     
         2 . The method according to  claim 1 , wherein said RNAi agent is an interfering ribonucleic acid.  
     
     
         3 . The method according to  claim 2 , wherein said interfering ribonucleic acid is a siRNA.  
     
     
         4 . The method according to  claim 2 , wherein said interfering ribonucleic acid is a shRNA.  
     
     
         5 . The method according to  claim 1 , wherein said RNAi agent is a transcription template of an interfering ribonucleic acid.  
     
     
         6 . The method according to  claim 5 , wherein said transcription template is a deoxyribonucleic acid.  
     
     
         7 . The method according to  claim 6 , wherein said deoxyribonucleic acid encodes a shRNA.  
     
     
         8 . The method according to  claim 1 , wherein said non-embryonic mammal is an adult.  
     
     
         9 . The method according to  claim 8 , wherein said non-embryonic mammal is a juvenile.  
     
     
         10 . The method according to  claim 1 , wherein said RNAi agent is hydrodynamically administered to said non-embryonic mammal.  
     
     
         11 . The method according to  claim 10 , wherein said RNAi agent is hydrodynamically administered to said non-embryonic mammal in conjunction with an RNAse inhibitor.  
     
     
         12 . A pharmaceutical preparation comprising an RNAi agent in a pharmaceutically acceptable delivery vehicle.  
     
     
         13 . The pharmaceutical preparation according to  claim 12 , wherein said preparation further comprises an RNAse inhibitor.  
     
     
         14 . A kit for use in practicing the method of  claim 1 , said kit comprising: 
 (a) a pharmaceutical preparation comprising an RNAi agent in a pharmaceutically acceptable delivery vehicle; and    (b) instructions for practicing the method of  claim 1 .    
     
     
         15 . The kit according to  claim 14 , wherein said kit further comprises an RNAse inhibitor.  
     
     
         16 . A non-embryonic non-human animal comprising an RNAi agent.  
     
     
         17 . A non-embryonic non-human animal produced according to the method of  claim 1 .  
     
     
         18 . A method for introducing a ribonucleic acid into a target cell of a vascularized multi-cellular organism, said method comprising: 
 administering said ribonucleic acid as a naked ribonucleic acid into the vascular system of said organism to introduce said ribonucleic acid into said target cell of said vascularized multi-cellular organism.    
     
     
         19 . The method according to  claim 18 , wherein said administering is intravenous.  
     
     
         20 . The method according to  claim 18 , wherein said vascularized multi-cellular organism is a mammal.  
     
     
         21 . The method according to  claim 18 , wherein said target cell is a hepatic cell.  
     
     
         22 . The method according to  claim 18 , wherein said method further comprises administering an RNAse inhibitor.  
     
     
         23 . The method according to  claim 18 , wherein said method further comprises administering a competitor ribonucleic acid to said organism.  
     
     
         24 . A method for introducing a nucleic acid into a target cell of a vascularized multi-cellular organism, said method comprising: 
 hydrodynamically administering to said vascularized multi-cellular organism an aqueous formulation comprising said nucleic acid as a naked nucleic acid and an RNase inhibitor to introduce said nucleic acid into said target cell.    
     
     
         25 . The method according to  claim 24 , wherein said nucleic acid is a deoxyribonucleic acid.  
     
     
         26 . The method according to  claim 24 , wherein said nucleic acid is ribonucleic acid.  
     
     
         27 . The method according to  claim 24 , wherein said aqueous formulation further comprises competitor ribonucleic acid.  
     
     
         28 . A kit for use in delivering a nucleic acid to a target cell of a vascularized multi-cellular organism, said kit comprising: 
 said nucleic acid present as a naked nucleic acid; and    an RNase inhibitor;    
     
     
         29 . The kit according to  claim 28 , wherein said nucleic acid is a deoxyribonucleic acid.  
     
     
         30 . The kit according to  claim 28 , wherein said nucleic acid is a ribonucleic acid.  
     
     
         31 . The kit according to  claim 28 , wherein said kit further comprises instructions for introducing said formulation into the vascular system a multi-cellular organism.  
     
     
         32 . The kit according to  claim 28 , wherein said kit further comprises a competitor ribonucleic acid.  
     
     
         33 . The kit according to  claim 28 , wherein said kit further comprises a means for vascularly administrating said aqueous formulation.  
     
     
         34 . A method of determining the activity of an RNA virus candidate modulatory agent, said method comprising: 
 (1) hydrodynamically administering to a vascularized multicellular animal: 
 (a) a nucleic acid construct that includes either: 
 (i) a RNA molecule that includes at least one regulatory element of said RNA virus operably linked to a reporter element; or  
 (ii) a DNA molecule capable of being transcribed into said RNA molecule; and  
 
 (b) said candidate modulatory agent; and  
   (2) observing the effect of said modulatory agent on the activity of said construct is to determine the activity of said candidate modulatory agent.    
     
     
         35 . The method according to  claim 34 , wherein said nucleic acid method further comprises hydrodynamically administering an RNAse inhibitor.  
     
     
         36 . The method according to  claim 34 , wherein said method further comprising hydrodynamically administering a competitor RNA.  
     
     
         37 . The method according to  claim 34 , wherein said RNA virus is HCV.