Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tighten gut mucosal barrier

Abstract

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of treating or preventing autoimmune disorders, inhibiting inflammatory mechanisms in the gut, and/or tightening gut mucosal barrier function are disclosed.

1 . A genetically engineered bacterium comprising:
 a) one or more non-native copies of a first gene that encodes a transcription factor protein that is regulated by a reactive nitrogen species (RNS), wherein the expression of the gene is operatively linked to a promoter; and   b) one or more of:
 i. a second gene encoding a non-native, anti-inflammation molecule; 
 ii. a second gene encoding a non-native gut barrier function enhancer molecule; and 
 iii. a gene cassette encoding a biosynthetic pathway, wherein the final product of the biosynthetic pathway is an anti-inflammation and/or a barrier function enhancer molecule, wherein 
   
       the expression of the second gene or gene cassette in b) is controlled by a tunable regulatory region heterologous to the gene or gene cassette, wherein induction of the tunable regulatory region is directly or indirectly controlled by the transcription factor. 
     
     
         2 . The bacterium of  claim 1 , wherein the transcription factor is a transcription activator. 
     
     
         3 . The bacterium of  claim 2 , wherein induction of the tunable regulatory region is directly controlled by the transcription activator. 
     
     
         4 . The bacterium of  claim 1 , wherein the transcription factor is a transcription repressor. 
     
     
         5 . The bacterium of  claim 4 , wherein derepression of the tunable regulatory region is directly controlled by the transcription repressor. 
     
     
         6 . The bacterium of  claim 1 , wherein:
 the transcription factor is a first transcription repressor;   the bacterium further comprises a gene encoding a second transcription repressor, wherein expression of the second transcription repressor is repressed by the first repressor; and   the tunable regulatory region is repressed by the second transcription repressor.   
     
     
         7 . The bacterium of  claim 1 , wherein at least one of the one or more non-native copies of the gene that encodes the transcription factor is located on a plasmid in the bacterium. 
     
     
         8 . The bacterium of  claim 1 , wherein at least one of the one or more non-native copies of the gene that encodes the transcription factor is located on a chromosome in the bacterium. 
     
     
         9 . The bacterium of  claim 1 , wherein the promoter that controls expression of at least one of the one or more non-native copies of the gene that encodes the transcription factor is a constitutive promoter. 
     
     
         10 . The bacterium of  claim 1 , wherein the promoter that controls expression of at least one of the one or more non-native copies of the gene that encodes the transcription factor is an inducible promoter. 
     
     
         11 . The bacterium of  claim 1 , wherein the gene encoding the anti-inflammation molecule, the gut barrier enhancer molecule, or the gene cassette encoding the biosynthetic pathway is located on a plasmid in the bacterium. 
     
     
         12 . The bacterium of  claim 1 , wherein the gene encoding the anti-inflammation molecule, the gut barrier enhancer molecule, or the gene cassette encoding the biosynthetic pathway is located on a chromosome in the bacterium. 
     
     
         13 . The bacterium of  claim 1 , wherein the gene that encodes the transcription factor protein is NsrR. 
     
     
         14 . The bacterium of  claim 1 , wherein the regulatory region that controls expression of the anti-inflammation molecule, gut barrier enhancer molecule, or the biosynthetic pathway is selected from a native or a modified functional form of any one of norB, aniA, nsrR, hmpA, ytfE, ygbA, hcp, hcr, nrfA, aox. 
     
     
         15 . The bacterium of  claim 1 , wherein the anti-inflammation and/or gut barrier enhancer molecule is selected from propionate, butyrate, acetate, IL-10, IL-27, TGF-β2, TGF-β1, GLP-2, NAPEs, elafin, trefoil factor, and scFv, antisense RNA, siRNA, or shRNA directed against a pro-inflammatory molecule. 
     
     
         16 . The bacterium of  claim 1 , wherein the bacterium is a non-pathogenic bacterium. 
     
     
         17 . (canceled) 
     
     
         18 . The bacterium of  claim 16 , wherein the bacterium is selected from the group consisting of  Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus , and  Lactococcus.    
     
     
         19 . The bacterium of  claim 18 , wherein the bacterium is  Escherichia coli  strain Nissle. 
     
     
         20 . The bacterium of  claim 1 , wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut. 
     
     
         21 . The bacterium of  claim 20 , wherein mammalian gut is a human gut. 
     
     
         22 . The bacterium of  claim 20 , wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . A pharmaceutically acceptable composition comprising the bacterium of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         26 . (canceled) 
     
     
         27 . A method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, the composition of  claim 25 . 
     
     
         28 . A method of treating a disease or condition associated with gut inflammation and/or compromised gut barrier function, comprising the step of administering to a patient in need thereof, the composition of claim  25 . 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 27 , wherein the autoimmune disorder is selected from the group consisting of type 1 diabetes, asthma, multiple sclerosis, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, Crohn's disease, celiac disease, and ankylosing spondylitis. 
     
     
         31 . The method of  claim 28 , wherein the disease or disorder is selected from an inflammatory bowel disease and a diarrheal disease.