Delivery of polypeptide-encoding plasmid dna into the cytsol of macrophages by attenuated suicide bacteria

Abstract

The invention relates to the introduction of DNA or RNA sequences into a mammalian cell to achieve controlled expression of a polypeptide. It is therefore useful in gene therapy, vaccination, and any therapeutic situation in which a polypeptide should be administered to a host or cells of said host, as well as for the production of polypeptides by mammalian cells, e.g., in Culture or in transgenic animals.

What is claimed is:  
     
         1 . An attenuated invasive intracellular bacterium capable of infecting a mammalian host or host cell thereof, but having a decreased ability in intra- and intercellular movement in said host as compared to a wild type bacterium, transformed with 
 (a) a promoter activated when said bacterium is present in the cytosol of a host cell, operably linked to a structural gene or fragment thereof encoding a polypeptide which is lethal to the bacterium,    (b) a host cell-compatible promoter, operably linked to a structural gene or fragment thereof encoding a polypeptide which has therapeutic and/or prophylactic properties.    
     
     
         2 . An attenuated invasive bacterium of  claim 1 , wherein (a) and (b) can be on the same plasmid or different plasmids.  
     
     
         3 . An attenuated invasive bacterium of  claim 1 , wherein (a) can be integrated into the bacterial chromosome and (b) is on a plasmid.  
     
     
         4 . An attenuated invasive bacterium of  claim 1 , wherein the bacterium is a species of Listeria, Salmonella, Renibacterium or Yerisina.  
     
     
         5 . An attenuated invasive bacterium of  claim 1 , wherein the bacterium is a  Listeria monocytogenes.    
     
     
         6 . An attenuated invasive bacterium of  claim 1 , wherein the bacterium lacks the lecithinase operon.  
     
     
         7 . An attenuated invasive bacterium of  claim 1 , wherein the polypeptide which is lethal to the bacterium is a bacteriophage lysin.  
     
     
         8 . An attenuated invasive bacterium of  claim 1 , wherein the promoter in (a) is the Listeria promoter P actA .  
     
     
         9 . An attenuated invasive bacterium of  claim 1 , wherein the host cell-compatible promoter in (b) is promoter P CMV .  
     
     
         10 . An attenuated invasive bacterium of  claim 1 , wherein the polypeptide which is lethal to the bacterium causes autolysis of the bacterium in the cytosol of said host cell.  
     
     
         11 . An attenuated invasive bacterium of  claim 1 , wherein the polypeptide which is lethal to the bacterium is Lys 118.  
     
     
         12 . A plasmid comprising (a) a promoter activated when it is present in an invasive bacterium which is in the cytosol of a mammalian host cell, operably linked to a structural gene encoding a protein which is lethal to the bacterium.  
     
     
         13 . A plasmid of  claim 12 , further comprising (b) a mammalian host cell-compatible promoter, operably linked to a structural gene encoding a protein which is an antigen.  
     
     
         14 . A plasmid of  claim 12 , wherein the protein which is lethal to the bacterium is a bacteriophage lysin.  
     
     
         15 . A plasmid of  claim 12 , wherein the promoter in (a) is the Listeria promoter P actA .  
     
     
         16 . A plasmid of  claim 12 , wherein the host cell-compatible promoter in (b) is promoter P CMV .  
     
     
         17 . A plasmid of  claim 12 , wherein the protein which is lethal to the bacterium causes autolysis of the bacterium in the cytosol of said host cell.  
     
     
         18 . A genetic transformation vector for a mammalian host cell in vivo or in vitro, comprising an attenuated invasive intracellular bacterium capable of infecting a mammalian host or host cell thereof, but having a decreased ability in intra- and intercellular movement in said host as compared to a wild type bacterium, transformed with 
 (a) a promoter activated when said bacterium is present in the cytosol of a host cell, operably linked to a structural gene or figment thereof encoding a polypeptide which is lethal to the bacterium,    (b) a host cell-compatible promoter, operably linked to a structural gene or fragment thereof encoding a polypeptide which has therapeutic and/or prophylactic properties.    
     
     
         19 . A vector of  claim 18 , wherein the structural gene in (b) encodes an antigen.  
     
     
         20 . A vector of  claim 18 , wherein the structural gene in (b) encodes a therapeutic agent.  
     
     
         21 . A vector of  claim 18 , wherein the structural gene in (b) encodes a secretable protein product.  
     
     
         22 . A method of vaccination, comprising administering to a patient in need of vaccination an effective amount of a vaccine of  claim 1 .  
     
     
         23 . A method of  claim 22 , whereby the vaccine invades the patient's monocytes.  
     
     
         24 . A method of  claim 23 , wherein the monocytes are professional antigen presenting cells (APCs).  
     
     
         25 . A method of  claim 24 , wherein the antigen protein encoded in (b) is expressed and presented by the APCs and whereby an immune response is elicited.  
     
     
         26 . A method of  claim 25 , wherein the patient's cells which have been infected by the vaccine bacterium are eradicated by the patient's cellular immune system after the antigen is presented.  
     
     
         27 . A method of  claim 22 , wherein the route of administration to the patient is oral.  
     
     
         28 . A method of delivering an expressible structural gene to a mammalian host cell, comprising an attenuated invasive intracellular bacterium capable of infecting said mammalian host or host cell thereof, but having a decreased ability in intra- and intercellular movement in said host as compared to a wild type bacterium, transformed with 
 (a) a promoter activated when said bacterium is present in the cytosol of a host cell, operably linked to a structural gene or fragment thereof encoding a polypeptide which is lethal to the bacterium,    (b) a host cell-compatible promoter, operably linked to a structural gene or fragment thereof encoding a polypeptide which has therapeutic and/or prophylactic properties.    
     
     
         29 . A method of  claim 28 , wherein said structural gene encodes an antigen.