Minicell compositions and methods

Abstract

The invention provides compositions and methods for the production of achromosomal and anucleate cells useful for applications such as diagnositic and therapeutic uses, as well as research tools and agents for drug discovery.

What is claimed:  
     
         1 . A method for making minicells, comprising 
 (a) culturing a minicell-producing parent cell, wherein said parent cell comprises an expression construct, wherein said expression construct comprises a gene operably linked to expression sequences that are inducible and/or repressible, and wherein induction or repression of said gene causes or enhances the production of minicells; and    (b) separating said minicells from said parent cell, thereby generating a composition comprising minicells,    wherein an inducer or repressor is present within said parent cells during one or more steps and/or between two or more steps of said method.    
     
     
         2 . The method of  claim 1 , further comprising 
 (c) purifying said minicells from said composition.    
     
     
         3 . The method of  claim 1 , wherein said minicell is selected from the group consisting of a eubacterial minicell, a poroplast, a spheroplast and a protoplast.  
     
     
         4 . The method of  claim 1 , wherein said gene expresses a gene product that is a factor that is involved in or modulates DNA replication, cellular division, cellular partitioning, septation, transcription, translation, or protein folding.  
     
     
         5 . The method of  claim 1 , wherein said minicells are separated from said parent cells by a process selected from the group consisting of centrifugation, ultracentrifugation, density gradation, immunoaffinity and immunoprecipitation.  
     
     
         6 . The method of  claim 2 , wherein said minicell is a poroplast, said method further comprising 
 (d) treating said minicells with an agent, or incubating said minicells under a set of conditions, that degrades the outer membrane of said minicell.    
     
     
         7 . The method of  claim 6 , wherein said outer membrane is degraded by treatment with an agent selected from the group consisting of EDTA, EGTA, lactic acid, citric acid, gluconic acid, tartaric acid, polyethyleneimine, polycationic peptides, cationic leukocyte peptides, aminoglycosides, aminoglycosides, protamine, insect cecropins, reptilian magainins, polymers of basic amino acids, polymixin B, chloroform, nitrilotriacetic acid and sodium hexametaphosphate and/or by exposure to conditions selected from the group consisting of osmotic shock and insonation.  
     
     
         8 . The method of  claim 6 , further comprising removing one or more contaminants from said composition.  
     
     
         9 . The method of  claim 8 , wherein said contaminant is LPS or peptidoglycan.  
     
     
         10 . The method of  claim 9 , wherein said LPS is removed by contacting said composition to an agent that binds or degrades LPS.  
     
     
         11 . The method of  claim 1 , wherein said minicell-producing parent cell comprises a mutation in a gene required for lipopolysaccharide synthesis.  
     
     
         12 . The method of  claim 2 , wherein said minicell is a spheroplast, said method further comprising 
 (d) treating said minicells with an agent, or incubating said minicells under a set of conditions, that disrupts or degrades the outer membrane; and    (e) treating said minicells with an agent, or incubating said minicells under a set of conditions, that disrupts or degrades the cell wall.    
     
     
         13 . The method of  claim 12 , wherein said agent that disrupts or degrades the cell wall is a lysozyme, and said set of conditions that disrupts or degrades the cell wall is incubation in a hypertonic solution.  
     
     
         14 . The method of  claim 2 , wherein said minicell is a protoplast, said method further comprising 
 (d) treating said minicells with an agent, or incubating said minicells under a set of conditions, that disrupt or degrade the outer membrane;    (e) treating said minicells with an agent, or incubating said minicells under a set of conditions, that disrupts or degrades the cell wall, in order to generate a composition that comprises protoplasts; and    (f) purifying protoplasts from said composition.    
     
     
         15 . The method of  claim 2 , further comprising preparing a denuded minicell from said minicell.  
     
     
         16 . The method of  claim 2 , further comprising covalently or non-covalently linking one or more components of said minicell to a conjugated moiety.  
     
     
         17 . A method of preparing a L-form minicell comprising: 
 (a) culturing an L-form eubacterium, wherein said eubacterium comprises one or more of the following: 
 (i) an expression element that comprises a gene operably linked to expression sequences that are inducible and/or repressible, wherein induction or repression of said gene regulates the copy number of an episomal expression construct;  
 (ii) a mutation in an endogenous gene, wherein said mutation regulates the copy number of an episomal expression construct.  
 (iii) an expression element that comprises a gene operably linked to expression sequences that are inducible and/or repressible, wherein induction or repression of said gene causes or enhances the production of minicells; and  
 (iv) a mutation in an endogenous gene, wherein said mutation causes or enhances minicell production.  
   (b) culturing said L-form minicell-producing parent cell in media under conditions wherein minicells are produced; and    (c) separating said minicells from said parent cell, thereby generating a composition comprising L-form minicells,    wherein an inducer or repressor is present within said minicells during one or more steps and/or between two or more steps of said method.    
     
     
         18 . The method of  claim 17 , further comprising 
 (d) purifying said L-form minicells from said composition.