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