Methods for wound healing

Abstract

The invention relates to plasmids capable of expressing a protein targeting immune cells when transformed into a lactic acid bacterial cell, wherein the protein is chosen from the group consisting of murine and human CXCL12 1α; CXCL17 and Ym1. The invention further relates to lactic acid bacteria transformed with a said plasmid, as well as the use of said lactic acid bacteria for wound healing in humans and animals.

1 . A plasmid which is capable of expressing a protein in lactic acid bacteria, wherein the said protein is selected from the group consisting of CXCL12, CXCL17 and Ym1. 
     
     
         2 . The plasmid of  claim 1 , wherein said plasmid comprises a nucleotide sequence encoding a protein selected from:
 (i) murine CXCL12-1α having an amino acid sequence as shown in SEQ ID NO: 3 or 2, or an amino acid sequence with at least 80% sequence identity thereto;   (ii) human CXCL12-1α having an amino acid sequence as shown in SEQ ID NO: 6 or 5, or an amino acid sequence with at least 80% sequence identity thereto;   (iii) murine CXCL17 having an amino acid sequence as shown in SEQ ID NO: 9 or 8, or an amino acid sequence with at least 80% sequence identity thereto;   (iv) human CXCL17 having an amino acid sequence as shown in SEQ ID NO: 12 or 11, or an amino acid sequence with at least 80% sequence identity thereto;   (v) murine Ym1 having an amino acid sequence as shown in SEQ ID NO: 15 or 14, or an amino acid sequence with at least 80% sequence identity thereto; and   (vi) human Ym1 as shown in SEQ ID NO: 18 or 17 or an amino acid sequence with at least 80% sequence identity thereto.   
     
     
         3 . The plasmid according to  claim 1 , wherein the plasmid comprises one or more regulatory sequences which permit expression in lactic acid bacteria, wherein the regulatory sequences are obtained or derived from lactic acid bacteria. 
     
     
         4 . The plasmid according to  claim 1 , wherein expression of said protein is regulatable. 
     
     
         5 . The plasmid according to  claim 1 , wherein the plasmid comprises one or more nucleotide sequences encoding one or more of said proteins under the control of an inducible promoter. 
     
     
         6 . The plasmid according to  claim 1 , wherein the plasmid comprises an inducible promoter and regulatory elements from the nisin regulon, the sakacin A regulon or the sakacin P regulon of a lactic acid bacterium. 
     
     
         7 . The plasmid according to  claim 5 , wherein the inducible promoter is the PorfX promoter from the sakacin P regulon. 
     
     
         8 . The plasmid according to  claim 1 , which is derived from the plasmid designated pSIP411. 
     
     
         9 . The recombinant plasmid according to  claim 1 , wherein the nucleotide sequence encoding the protein is codon-optimised for expression in lactic acid bacteria. 
     
     
         10 . The plasmid according to  claim 1 , which comprises one or more nucleotide sequences selected from the group consisting of: a nucleotide sequence comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16, or a nucleotide sequence having at least 80% sequence identity to any aforesaid sequence. 
     
     
         11 . A lactic acid bacterial strain transformed with the plasmid according to  claim 1 . 
     
     
         12 . The bacterial strain according to  claim 11 , which is a  Lactobacillus  strain. 
     
     
         13 . The bacterial strain according to  claim 12 , which is a strain of  Lactobacillus reuteri.    
     
     
         14 . A wound dressing comprising the bacterial strain according to  claim 11 . 
     
     
         15 . A pharmaceutical composition comprising the bacterial strain according to  claim 11 . 
     
     
         16 . The plasmid of  claim 1  or a bacterial strain transformed with the plasmid for use in therapy. 
     
     
         17 . The plasmid of  claim 1  or a bacterial strain transformed with the plasmid for use in wound healing in a human or animal subject. 
     
     
         18 . The plasmid of  claim 1  or a bacterial strain transformed with the plasmid for use in cutaneous or mucosal wound healing in a human or animal subject. 
     
     
         19 . A kit for healing wounds, said kit comprising:
 (i) lactic acid bacteria comprising the plasmid of  claim 1 , wherein said plasmid comprises a nucleotide sequence encoding a said protein under the control of an inducible promoter capable of expressing the protein in lactic acid bacteria; and   (ii) an inducer for the promoter.   
     
     
         20 . The kit of  claim 19 , being a pharmaceutical product comprising;
 (i) lactic acid bacteria comprising the plasmid of  claim 1 , wherein said plasmid comprises a nucleotide sequence encoding a said protein under the control of an inducible promoter capable of expressing the protein in lactic acid bacteria; and   (ii) an inducer for the promoter.   as a combined preparation for separate, sequential or simultaneous use in wound healing.   
     
     
         21 . A medical device comprising the bacterial strain according to  claim 11 . 
     
     
         22 . The bacterial strain of  claim 11  wherein the bacteria are freeze-dried. 
     
     
         23 . A kit for healing wounds, said kit comprising:
 the bacterial strain of  claim 22 , wherein the plasmid comprises a nucleotide sequence encoding the protein under the control of an inducible promoter capable of expressing the protein in lactic acid bacteria;   an inducer for the promoter; and   a liquid for resuspending the freeze-dried bacteria.   
     
     
         24 . The kit of  claim 23 , wherein the liquid comprises the inducer. 
     
     
         25 . The kit of  claim 19 , wherein the kit comprises a wound dressing comprising the bacteria. 
     
     
         26 . The kit of  claim 19 , wherein the kit further comprises a wound dressing.