Intracellular delivery vehicles

Abstract

The invention provides methods and compositions relating to intracellular delivering of agents to eukaryotic cells. The compositions include microbial delivery vehicles such as nonvirulent bacteria comprising a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter and a second gene encoding a different foreign agent. The foreign agent may be a nucleic acid or protein, and is frequently bioactive in and therapeutic to the target eukaryote. In addition, the invention provides eukaryotic cells comprising the subject nonvirulent bacteria and nonhuman eukaryotic host organisms comprising such cells.

What is claimed is:  
     
         1 . A nonvirulent bacterium comprising a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter and a second gene encoding a different foreign agent.  
     
     
         2 . The bacterium of  claim 1 , wherein the cytolysin is absent a functional signal sequence.  
     
     
         3 . The bacterium of  claim 1 , wherein the cytolysin is listeriolysin.  
     
     
         4 . The bacterium of  claim 1 , wherein the bacterium further comprises a third gene encoding an invasin and a fourth gene encoding an autolysin.  
     
     
         5 . The bacterium of  claim 1 , wherein the bacterium is a laboratory strain of  E. coli.    
     
     
         6 . The bacterium of  claim 1 , wherein the foreign agent is therapeutic to a eukaryote.  
     
     
         7 . A eukaryotic cell comprising the nonvirulent bacterium of  claim 1  further comprising the foreign cytolysin.  
     
     
         8 . The cell of  claim 7 , wherein the cell is phagocytic, pathogenic or diseased cell.  
     
     
         9 . A method for introducing a foreign agent into a eukaryotic cell comprising the step of contacting the cell with the bacterium of  claim 1  under conditions whereby the agent enters the cell.  
     
     
         10 . The method of  claim 9 , wherein the bacterium is endocytosed into a vacuole of the cell, the bacterium undergoes lysis and the cytolysin mediates transfer of the agent from the vacuole to the cytosol of the cell.  
     
     
         11 . A method of generating an immune response to a foreign antigenic agent, comprising the step of introducing the foreign antigenic agent into a cell of an patient by contacting the cell with a bacterium under conditions whereby the agent enters the cell, 
 said bacterium comprising a first gene encoding a nonsecreted foreign functional cytolysin operably linked to a heterologous promoter which expresses the cytolysin in the bacterium, and a second gene encoding the foreign antigenic agent, which is other than the cytolysin.    
     
     
         12 . A method of generating an physiological response to a therapeutic agent, comprising the step of introducing the therapeutic agent into a cell of an patient by contacting the cell with a bacterium under conditions whereby the agent enters the cell, 
 said bacterium comprising a first gene encoding a nonsecreted foreign functional cytolysin operably linked to a heterologous promoter which expresses the cytolysin in the bacterium, and a second gene encoding the therapeutic agent, which is other than the cytolysin.