Regulated expression of recombinant proteins using RNA viruses

Abstract

The present invention describes cells and constructs for a regulated viral (e.g. alphavirus) expression system, where gene expression is controlled by controlling expression of replicases or nonstructural proteins and/or controlling the amount of such proteins introduced in a cell, which in turn regulates RNA replication and subsequently gene expression. Particularly, this system takes advantage of the high level expression of the alphavirus systems for recombinant protein production and allows for large scale applications without biosafety concerns.

What is claimed is:  
     
         1 . A method for controlling viral RNA replication or gene expression in a cell, said method comprising controlling expression of said one or more genes encoding one or more viral replicases or nonstructural proteins in said cell and/or introducing one or more of said replicases or nonstructural proteins in said cell.  
     
     
         2 . The method according to  claim 1 , wherein one or more of said genes is under control of an inducible promoter.  
     
     
         3 . The method according to  claim 1 , wherein said method controls alphavirus RNA replication.  
     
     
         4 . The method according to  claim 1 , wherein said one or more genes is under control of a constitutive promoter.  
     
     
         5 . The method according to  claim 1 , wherein said method further comprises controlling expression of one or more genes of interest.  
     
     
         6 . The method according to  claim 5 , wherein one or more of said genes of interest are under control of a viral promoter.  
     
     
         7 . The method according to  claim 1 , wherein one or more of said replicases or nonstructural proteins are introduced into said cell by using one or more cationic lipids.  
     
     
         8 . The method according to  claim 1 , wherein said one or more replicases or nonstructural proteins are introduced into said cell by fusing said replicases or proteins with a peptide or protein sequence having a transport function such that said replicase protein is transported into said cell.  
     
     
         9 . The method according to  claim 3 , wherein said alphavirus is selected from the group consisting of Semliki Forest Virus, Sinbis Virus, Venezuelan Equine Encephalitis Virus, Eastern Equine Encephalitis Virus.  
     
     
         10 . The method according to  claim 1 , wherein said replicases or nonstructural proteins are selected from the group consisting of nonstructural proteins 1, 2, 3 and 4 and combinations thereof.  
     
     
         11 . A nucleic acid molecule comprising one or more genes encoding one or more viral replicases or nonstructural proteins under control of one or more inducible promoters.  
     
     
         12 . The nucleic acid molecule according to  claim 11 , wherein said one or more genes encode for nonstructural proteins 1, 2, 3 or 4 or combination thereof.  
     
     
         13 . The nucleic acid molecule according to  claim 11 , wherein said viral replicases or nonstructural proteins are derived from an alphavirus.  
     
     
         14 . The nucleic acid molecule according to  claim 13 , wherein said alphavirus is selected from the group consisting of Semliki Forest Virus, Sinbis Virus, Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus.  
     
     
         15 . The nucleic acid molecule according to  claim 11 , wherein said molecule is a vector.  
     
     
         16 . The nucleic acid molecule according to  claim 11 , wherein one or more of said genes is under control of one or more constitutive promoters.  
     
     
         17 . The nucleic acid molecule according to  claim 11 , further comprising one or more genes of interest under control of a viral promoter.  
     
     
         18 . The nucleic acid molecule according to  claim 17 , wherein said viral promoter is an alphavirus recognized promoter.  
     
     
         19 . The nucleic acid molecule according to  claim 18 , wherein said alphavirus is selected from the group consisting of Semliki Forest Virus, Sinbis Virus, Venezuelan Equine Encephalitis Virus, Eastern Equine Encephalitis Virus.  
     
     
         20 . A host cell comprising the nucleic acid molecule according to  claim 11 .  
     
     
         21 . A host cell comprising the nucleic acid molecule according to  claim 17 .  
     
     
         22 . A pharmaceutical composition comprising a cell of  claim 20  and a pharmaceutical carrier.  
     
     
         23 . A pharmaceutical composition comprising a cell of  claim 21  and a pharmaceutical carrier.  
     
     
         24 . A method for controlling expression of a gene of interest in a cell comprising providing a factor or drug which inhibits or prevents viral RNA replication in said cell.  
     
     
         25 . The method of  claim 24 , wherein said factor is MxA.