Bacteria Engineered to Treat Diseases that Benefit from Reduced Gut Inflammation and/or Tightened Gut Mucosal Barrier

• US 20180312851A1
• Filed: 09/08/2016
• Published: 11/01/2018
• Est. Priority Date: 10/30/2015
• Status: Active Grant

• Current Assignee: Synlogic Operating Company, Inc. (Synlogic, Inc.)
• Original Assignee: Synlogic Operating Company, Inc. (Synlogic, Inc.)
• Inventors: Falb, Dean, Isabella, Vincent M., Kotula, Jonathan W., Miller, Paul F., Millet, Yves, Fisher, Adam
• Granted Patent: US 11,685,925 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2035/115 Probiotics
• CPC Class Codes: A61K 31/19 Carboxylic acids, e.g. valproic acid salicylic acid A61K31/60
• CPC Class Codes: A61K 31/198 Alpha-amino acids, e.g. alanine or edetic acid [EDTA] betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00
• CPC Class Codes: A61K 35/741 Probiotics probiotic yeast, e.g. saccharomyces A61K36/06
• CPC Class Codes: A61K 38/20 Interleukins [IL]
• CPC Class Codes: A61K 38/2013 IL-2
• CPC Class Codes: A61K 38/2066 IL-10
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/446 Superoxide dismutase (1.15)
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1217 Phosphotransferases with a carboxyl group as acceptor (2.7.2)
• CPC Class Codes: C12Y 103/08001 Butyryl-CoA dehydrogenase (1.3.8.1), i.e. short chain acyl-CoA dehydrogenase
• CPC Class Codes: C12Y 115/01001 Superoxide dismutase (1.15.1.1)
• CPC Class Codes: C12Y 207/02007 Butyrate kinase (2.7.2.7)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A bacterium comprising at least one gene or gene cassette encoding one or more non-native gut barrier function enhancer molecule(s) or biosynthetic pathway(s) for producing a gut barrier function enhancer molecule, wherein at least one of the gut barrier enhancer molecule(s) is selected from an aryl hydrocarbon receptor (AHR) agonist, Pregnane X receptor (PXR) agonist, histone deacetylase (HDAC) inhibitor, GPR41 activator, GPR43 activator, and GPR109A, activator and wherein the at least one gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut.

2. The bacterium of claim 1, wherein the bacterium further comprises at least one gene or gene cassette encoding one or more non-native anti-inflammation molecule(s).

3. The bacterium of claim 1 or claim 2, wherein the promoter is induced under low-oxygen or anaerobic conditions.

4. The bacterium of claim 3, wherein the promoter is a FNR-responsive promoter, an ANR-responsive promoter, or a DNR-responsive promoter.

5. The bacterium of claim 4, wherein the promoter is a FNR-responsive promoter.

6. The bacterium of claim 1 or claim 2, wherein the promoter is induced by the presence of reactive nitrogen species.

7. The bacterium of claim 6, wherein the promoter is a NsrR-responsive promoter, NorR-responsive promoter, or a DNR-responsive promoter.

8. The bacterium of claim 1 or claim 2, wherein the promoter is induced by the presence of reactive oxygen species.

9. The bacterium of claim 8, wherein the promoter is a OxyR-responsive promoter, PerR-responsive promoter, OhrR-responsive promoter, SoxR-responsive promoter, or a RosR-responsive promoter.

10. The bacterium of any one of claims 1-9, wherein the gene and/or gene cassette is located on a chromosome in the bacterium.

11. The bacterium of any one of claims 1-9, wherein the at least one gene and/or gene cassette is located on a plasmid in the bacterium.

12. The bacterium of any one of claims 1-11, wherein the aryl hydrocarbon receptor (AHR) agonist is selected from indole, indole-3 aldehyde, indole-3 propionic acid, indole-3 acetate, kynurenine, 6-formylindolcarbazole, and kynurenic acid.

13. The bacterium of any one of claims 1-11, wherein the Pregnane X receptor (PXR) agonist is indole 3-propionic acid (IPA).

14. The bacterium of any one of claims 1-11, wherein the histone deacetylase (HDAC) inhibitor is butyrate.

15. The bacterium of any one of claims 1-11, wherein the GPR41 activator is selected from butyrate, propionate, and acetate.

16. The bacterium of any one of claims 1-11, wherein the GPR43 activator is selected from butyrate, propionate, and acetate.

17. The bacterium of any one of claims 1-11, wherein the GPR109A activator is selected from butyrate, kynurenic acid, and niacin.

18. The bacterium of any one of claims 1-17, wherein the anti-inflammation and/or gut barrier enhancer molecule is selected from a short-chain fatty acid, propionate, butyrate, acetate, IL-10 (human), IL-10 (viral), IL-2, IL-19, IL-20, IL-22, IL-24, IL-27, TGF-R2, TGF-R1, GLP-2, elafin, trefoil factor, superoxide dismutase (SOD), GLP-2, GLP-2 analog, GLP-1, N-acylphosphatidylethanolamines (NAPES), melatonin, tryptophan, tryptophan metabolite, PGD₂, kynurenic acid, indole, and indole metabolite.

19. The bacterium of claim 18, wherein the anti-inflammation and/or gut barrier enhancer molecule is a short-chain fatty acid.

20. The bacterium of claim 19, wherein the anti-inflammation and/or gut barrier enhancer molecule is selected from propionate, butyrate, and acetate.

21. The bacterium of claim 18, wherein the anti-inflammation and/or gut barrier enhancer molecule is selected from IL-10, IL-22, and GLP-2.

22. The bacterium of claim 19, further comprising an anti-inflammation and/or gut barrier enhancer molecule selected from IL-10, IL-22, and GLP-2.

23. The bacterium of any one of claims 1-17, wherein the anti-inflammation and/or gut barrier enhancer molecule is selected from a scFv, antisense RNA, siRNA, and shRNA directed against a pro-inflammatory molecule selected from TNF-α

, IFN-γ

, IL-1β

, IL-6, IL-8, IL-17, CXCL-8, and CCL2.

24. The bacterium of claim 23, further comprising one or more anti-inflammation and/or gut barrier enhancer molecule(s) selected from butyrate, propionate, acetate, IL-10, IL-22, and GLP-2.

25. The bacterium of any one of claims 1-24, wherein the bacterium has one or more native genes that have been mutated or deleted, wherein the native genes are selected from lpp, pal, tolA, nlpl, degP, and ompT.

26. The bacterium of any one of claims 1-25, wherein the anti-inflammation and/or gut barrier enhancer molecule comprises an N-terminal secretion tag.

27. The bacterium of claim 26, wherein the N-terminal secretion tag facilitates sec-dependent secretion of the anti-inflammation and/or gut barrier enhancer molecule.

28. The bacterium of claim 26, wherein the N-terminal secretion tag facilitates tat-dependent secretion of the anti-inflammation and/or gut barrier enhancer molecule.

29. The bacterium of claim 27, wherein the N-terminal secretion tag is selected from PhoA, OmpF, OmpA, and CvaC.

30. The bacterium of claim 28, wherein the N-terminal secretion tag is selected from TorA, FdnG, and DmsA.

31. The bacterium of any of claims 1-24, wherein the gene(s) or gene cassette(s) encoding the anti-inflammation and/or gut barrier enhancer molecule(s) comprises a 5′

-UTR sequence derived from the native fliC gene.

32. The bacterium of claim 31, wherein the gene(s) or gene cassette(s) encoding the anti-inflammation and/or gut barrier enhancer molecule(s) further comprise gene sequence encoding at least the first 20 amino acids of FliC.

33. The bacterium of any one of claims 1-32, wherein the bacterium is a probiotic bacterium.

34. The bacterium of claim 33, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.

35. The bacterium of claim 34, wherein the bacterium is Escherichia coli strain Nissle.

36. The bacterium of any one of claims 1-35, wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.

37. The bacterium of claim 36, wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.

38. A pharmaceutically acceptable composition comprising the bacterium of any one of claims 1-37;

and a pharmaceutically acceptable carrier.

39. The composition of claim 38 formulated for oral or rectal administration.

40. A method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, the composition of any one of claim 38 or 39.

41. 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 any one of claim 38 or 39.

42. The method of claim 40, 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 (UP), 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.

43. The method of claim 42, wherein the autoimmune disorder is selected from the group consisting of type 1 diabetes, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, celiac disease, and ankylosing spondylitis.

44. The method of claim 41, wherein the disease or condition is selected from an inflammatory bowel disease, including Crohn'"'"'s disease and ulcerative colitis, and a diarrheal disease.