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) at least one non-native copy of a first gene that encodes a transcription factor protein that is regulated by a reactive nitrogen species (RNS), wherein the first gene is operatively linked to a promoter; and   b) at least one 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; 
 iii. a gene cassette encoding a biosynthetic pathway, wherein a final product of the biosynthetic pathway is an anti-inflammation molecule; 
 iv. a gene cassette encoding a biosynthetic pathway, wherein a final product of the biosynthetic pathway is a gut barrier function enhancer molecule, wherein 
   
       the second gene or gene cassette in b) is expressed under the control of 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 third 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 nitric oxide sensing repressor NsrR. 
     
     
         14 . The bacterium of  claim 1 , wherein the tunable regulatory region that controls expression of the anti-inflammation molecule, the gut barrier enhancer molecule, or the biosynthetic pathway is selected from a native or a modified functional form of a regulatory region from any one of nitric oxide reductase (norB), aniA, nsrR, hmpA, ytfE, ygbA, hcp, hcr, nrfA, and alternative oxidase (aox). 
     
     
         15 . The bacterium of  claim 1 , wherein the molecule of b) is selected from propionate, butyrate, acetate, interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor ß2 (TGF-ß2), transforming growth factor ß1 (TGF-ß1), glucagon-like peptide (GLP-2), N-acylphosphatidylethanolamines (NAPEs), elafin, trefoil factor, and single-chain variable fragment (scFv), antisense RNA, short interfering RNA (siRNA), or short hairpin RNA (shRNA) directed against a pro-inflammatory molecule. 
     
     
         16 . The bacterium of  claim 1 , wherein the bacterium is a non-pathogenic bacterium. 
     
     
         17 . The bacterium of  claim 16 , wherein the bacterium is a probiotic bacterium. 
     
     
         18 . The bacterium of  claim 17 , 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 . The bacterium of  claim 1 , wherein the bacterium is further engineered to harbor an additional gene coding for a substance toxic to the bacterium, wherein the additional gene is under the control of a promoter that is directly or indirectly induced by an environmental factor not naturally present in a mammalian gut. 
     
     
         24 . The bacterium of  claim 1 , wherein the bacterium is further engineered to harbor an additional gene coding for a substance toxic to the bacterium, wherein the additional gene is under the control of a promoter that is indirectly induced by the transcription factor, and wherein the expression of the toxic substance is delayed in time as compared to the expression of the anti-inflammation molecule, the gut barrier enhancer molecule or the gene cassette encoding the biosynthetic pathway. 
     
     
         25 . A pharmaceutically acceptable composition comprising the bacterium of  claim 1 ; and a pharmaceutically acceptable carrier. 
     
     
         26 . The composition of  claim 25  formulated for oral or rectal administration. 
     
     
         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 . The method of  claim 27 , wherein the autoimmune disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, Axonal & neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with  Streptococcus ), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis. 
     
     
         30 . The method of  claim 29 , wherein the autoimmune disorder is selected from the group consisting of type 1 diabetes, asthma, multiple sclerosis, Crohn's disease, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, 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.