Polycistronic expression system for bacteria

Abstract

The invention relates to polycistronic expression in gram-positive bacterium and in particular concerns polycistronic expression units comprising one or more gene endogenous to the gram-positive bacterium transcriptionally coupled to one or more genes exogenous to the bacterium.

1 - 21 . (canceled) 
     
     
         22 . A method comprising:
 i) administering to a human subject or an animal subject a recombinant nucleic acid comprising a polycistronic expression unit, wherein said recombinant nucleic acid is present in a Gram-positive bacterium, wherein said polycistronic expression unit comprises:   a gene endogenous to said Gram-positive bacterium, and   one or more genes exogenous to said Gram-positive bacterium, which encode a therapeutic product, wherein said endogenous gene and said one or more exogenous genes are transcriptionally controlled by a promoter endogenous to said Gram-positive bacterium, and wherein said promoter is a ribosomal gene promoter, a glycolysis gene promoter, or a usp45 gene promoter, and   ii) delivering said therapeutic product to said human subject or said animal subject by said administration.   
     
     
         23 . The method of  claim 22 , wherein said promoter is selected from the group consisting of: the promoter of eno, usp45, gap, pyk, rpmB, and rplS of said Gram-positive bacterium. 
     
     
         24 . The method of  claim 22 , wherein said one or more exogenous genes are transcriptionally coupled to the 3′ end of said endogenous gene. 
     
     
         25 . The method of  claim 24 , wherein said one or more exogenous genes is the most 3′ gene of said polycistronic expression unit. 
     
     
         26 . The method of  claim 24 , wherein said endogenous gene and said one or more exogenous genes are transcriptionally coupled by one or more intergenic regions active in said Gram-positive bacterium. 
     
     
         27 . The method of  claim 26 , wherein the one or more intergenic regions are endogenous to said Gram-positive bacterium. 
     
     
         28 . The method of  claim 26 , wherein said one or more intergenic regions are preceding rplW, rplP, rpmD, rplB, rpsG, rpsE, rplN, rplM, rplE, or rplF. 
     
     
         29 . The method of  claim 22 , wherein said therapeutic product is an antigen for inducing immunity or immunotolerance, a non-vaccinogenic therapeutically active polypeptide, an antibody or a functional fragment thereof, a fusion protein, or a multimeric protein. 
     
     
         30 . The method of  claim 29 , wherein said functional fragment is an Fab. 
     
     
         31 . The method of  claim 29 , wherein one exogenous gene encodes a light chain (V L ) of an antibody or of a functional fragment thereof, and another exogenous gene encodes a heavy chain (V II ) of the antibody or of a functional fragment thereof. 
     
     
         32 . The method of  claim 31 , wherein the exogenous gene encoding said V L  or functional fragment thereof is transcriptionally coupled to the 3′ end of the exogenous gene encoding said V H  or functional fragment thereof. 
     
     
         33 . The method of  claim 22 , wherein said Gram-positive bacterium is a lactic acid bacterium or a  Bifidobacterium.    
     
     
         34 . The method of  claim 33 , wherein said lactic acid bacterium is a  Lactococcus , a  Lactobacillus , or an  Enterococcus.    
     
     
         35 . The method of  claim 34 , wherein said lactic acid bacterium is  Lactococcus lactis  or  Enterococcus faecium.