Inducible alphaviral gene expression system

Abstract

The present invention provides novel expression vectors which permit tight regulation of gene expression in eucaryotic cells. More specifically, the invention provides DNA vectors comprising nucleotide sequences that are transcribed to form RNA molecules which are then replicated by a temperature-sensitive replicase to form additional RNA molecules. The RNA molecules produced by replication contain a nucleotide sequence which may be translated to produce a protein of interest or which encode one or more untranslated RNA molecules. Also provided are methods for producing heterologous proteins and untranslated RNA molecules. Further provided are methods for administering heterologous proteins and untranslated RNA molecules to individuals. In addition, pharmaceutical compositions are provided comprising the DNA and RNA molecules of the invention and a pharmaceutically acceptable carrier.

1 - 74 . (canceled)  
     
     
         75 . A DNA molecule which encodes an RNA molecule comprising: 
 (a) at least one cis-acting sequence element,    (b) a first open reading frame which encodes a non-cytopathic temperature-sensitive alphaviral replicase, wherein non-cytopathicity and temperature sensitivity are conferred by one or more mutations in the genes encoding the nonstructural proteins of said replicase, and    (c) at least one second nucleotide sequence selected from the group consisting of: 
 (i) a second open reading frame encoding a protein, or portion thereof, wherein said second open reading frame is in a translatable format after one or more RNA-dependent RNA replication events;  
 (ii) a sequence complementary to all or part of the second open reading frame of (i); and  
 (iii) a sequence encoding an untranslated RNA molecule, or complement thereof;  
 wherein said second nucleotide sequence is operably linked to a promoter which is recognized by said non-cytopathic, temperature-sensitive alphaviral replicase.  
   
     
     
         76 . The DNA molecule of  claim 75 , which comprises one second nucleotide sequence.  
     
     
         77 . The DNA molecule of  claim 75 , wherein said second open reading frame is in a translatable format after one RNA-dependent RNA replication event.  
     
     
         78 . The DNA molecule of  claim 75 , wherein said second open reading frame is in a translatable format after three RNA-dependent RNA replication events.  
     
     
         79 . The DNA molecule of  claim 75 , wherein said alphaviral replicase is derived from a virus selected from the group consisting of: (a) Aura virus; (b) Bebaru virus; (c) Cabassou virus; (d) Chikungunya virus; (e) Eastern equine encephalomyelitis virus; (f) Fort Morgan virus; (g) Getah virus; (h) Kyzylagach virus; (i) Mayoaro virus; (j) Middleburg virus; (k) Mucambo virus; (l Ndumu virus; (m) Pixuna virus; (n) Semliki Forest Virus; (o) Tonate virus; (p) Triniti virus; (q) Una virus; (r) Western equine encephalomyelitis virus; (s) Whataroa virus; (t) Venezuelan equine encephalomyelitis virus (VEE); and (u) Ross River virus.  
     
     
         80 . The DNA molecule of  claim 75 , wherein said alphaviral replicase is derived from a Sindbis virus.  
     
     
         81 . The DNA molecule of  claim 75  which encodes an alphaviral replicase having replicase activity at 34° C. which is at least five fold lower than the replicase activity exhibited at 29° C.  
     
     
         82 . The DNA molecule of  claim 75 , wherein non-cytopathicity is conferred by one or more mutations in the nsP2 gene of said replicase.  
     
     
         83 . The DNA molecule of  claim 75 , wherein temperature sensitivity is conferred by one or more mutations in the nsP4 gene of said replicase.  
     
     
         84 . The DNA molecule of  claim 75 , wherein non-cytopathicity is conferred by one or more mutations in the nsP2 gene of said replicase, and temperature sensitivity is conferred by one or more mutations in the nsP4 gene of said replicase.  
     
     
         85 . The DNA molecule of  claim 75 , wherein the second open reading frame encodes a cytokine, a lymphokine, a tumor necrosis factor, an interferon, a toxic protein, or a prodrug converting enzyme.  
     
     
         86 . The DNA molecule of  claim 75 , wherein the second open reading frame encodes human erythropoietin or human β-interferon.  
     
     
         87 . A DNA vector system comprising one or more polynucleotides which encode RNA molecules, said RNA molecules comprising: 
 (a) at least one cis-acting sequence element,    (b) a first open reading frame having a nucleotide sequence encoding a non-cytopathic, temperature-sensitive alphaviral replicase, wherein non-cytopathicity and temperature sensitivity are conferred by one or more mutations in the genes encoding the nonstructural proteins of said replicase, and    (c) at least one second nucleotide sequence selected from the group consisting of: 
 (i) a second open reading frame encoding a protein, or portion thereof, wherein said second open reading frame is in a translatable format after one or more RNA-dependent RNA replication events;  
 (ii) a sequence complementary to all or part of the second open reading frame of (i); and  
 (iii) a sequence encoding an untranslated RNA molecule, or complement thereof;  
 wherein said second nucleotide sequence is operably linked to a promoter which is recognized by said non-cytopathic, temperature-sensitive alphaviral replicase.  
   
     
     
         88 . The DNA vector system of  claim 87 , wherein non-cytopathicity is conferred by one or more mutations in the nsP2 gene of said replicase, and temperature sensitivity is conferred by one or more mutations in the nsP4 gene of said replicase.  
     
     
         89 . The DNA vector system of  claim 87 , wherein said alphaviral replicase is derived from a Sindbis virus.  
     
     
         90 . A method for producing a protein or an untranslated RNA molecule in a recombinant host cell. comprising: 
 (a) introducing at least one DNA molecule of  claim 75  or at least one DNA vector system of  claim 87  into said host cells in vitro;    (b) culturing said host cells under conditions suitable for expression of said protein or untranslated RNA molecule; and    (c) recovering said protein or untranslated RNA molecule;    wherein said protein or untranslated RNA molecule is encoded by said DNA molecule or said DNA vector system.    
     
     
         91 . The method of  claim 90 , wherein said at least one DNA molecule or said at least one DNA vector system encodes a non-cytopathic, temperature-sensitive alphaviral replicase, wherein non-cytopathicity is conferred by one or more mutations in the nsP2 gene of said replicase.  
     
     
         92 . The method of  claim 90 , wherein said at least one DNA molecule or said at least one DNA vector system encodes a non-cytopathic, temperature-sensitive alphaviral replicase, wherein temperature sensitivity is conferred by one or more mutations in the nsP4 gene of said replicase.  
     
     
         93 . The method of  claim 92 , wherein said alphaviral replicase is derived from a Sindbis virus.  
     
     
         94 . The method of  claim 93 , wherein said protein is human erythropoietin or human β-interferon.