Engineered commensal bacteria and methods of use

Abstract

To provide compositions comprising populations of commensal bacteria isolated from a microbiome sample of a mammalian subject and engineered to express a heterologous polynucleotide, compositions comprising such engineered commensal bacteria and methods of use for delivering a therapeutic polypeptide to a mammal, e.g, by administering the engineered commensal / native bacteria.SOLUTION: Disclosed is a method of delivering a therapeutic polypeptide to a mammalian subject in need thereof, the method comprising: a) obtaining a microbiome sample comprising bacterial cells from a donating subject; b) isolating a bacterial cell from the microbiome sample, where the bacteria cell is from a bacterial strain that is commensal / native to the donating subject; c) culturing the isolated bacterial cell in vitro to yield a substantially homogeneous population of the isolated and cultured bacteria cell; d) transforming the bacterial cell population with one or more polynucleotides that are heterologous to the bacteria and / or the donating subject, where the one or more polynucleotides encode one or more therapeutic polypeptides; and e) administering or causing to be administered to a receiving subject at least a portion of the substantially homogeneous and transformed population of the isolated and cultured bacterial cells, where the administered bacterial cells are capable of colonizing permanently or long-term in or on the mammalian subject and expressing the one or more therapeutic polypeptides.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 486,068, filed April 17, 2017, which is expressly incorporated herein by reference in its entirety for all purposes.

[0002] Statement of government support This work was made with government support under DK102902 and DK114536 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Recent advances in sequencing, mass spectrometry, and bioinformatics are advancing our understanding of the role the microbiome plays in a host of physiological processes, including, but not limited to, metabolism, inflammation, behavior, and neurological diseases (Vuong et al., Annu Rev Neurosci. (2017) 40:21-49) [also cited in 25271724]. Linking physiological processes to the microbiome is conceptually exciting, but the field is still in its infancy. While studies have demonstrated strong associations between the microbiome and various physiological processes, few demonstrate clear mechanistic relationships. For example, the absolute abundance of analytes regulated by the gut microbiome, as well as signaling molecules from the gut epithelium, make it difficult to identify microbiome functions that may contribute to neurobehavioral processes. To develop a better mechanistic understanding and more effective microbiome-mediated therapies, various approaches focused on functional modulation of the gut microbiome are needed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent Publication No. 2016 / 0367701

Patent document 2

Patent Document 3

Non-licensed literature

[0005] [Non-licensed document 1] Vuongら, Annu Rev Neurosci. (2017) 40:21-49 [Non-licensed document 2] Rajilic-Stojanovic, FEMS Microbiol Rev. 2014;38:996-1047 [Non-licensed document 3] Human Microbiome Project Consortium in Nature. (2012) June 13;486(7402):207-14

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

Non-licensed Document 25

Non-licensed Document 26

Non-licensed Document 27

Non-licensed Document 28

Non-licensed Document 29

Non-licensed Document 30

Non-licensed Document 31

Non-licensed Document 32

Non-licensed Document 33

Non-licensed Document 34

Non-licensed Document 35

Non-licensed Document 36

Non-licensed Document 37

Non-licensed Document 38

Non-licensed Document 39

Non-licensed Document 40

Non-licensed Document 41

Non-licensed Document 42

Non-licensed Document 43

Non-licensed Document 44

[0006] In one aspect, a method for delivering a therapeutic polypeptide to a mammalian subject in need thereof is provided. In some embodiments, the method comprises: a) obtaining a microbiome sample comprising bacterial cells from a subject; b) isolating bacterial cells from the microbiome sample, wherein the isolated bacterial cells are derived from a symbiotic / native strain to the subject; c) culturing the isolated bacterial cells in vitro to obtain a substantially homogeneous population of isolated and cultured bacterial cells; d) transforming the substantially homogeneous population with one or more polynucleotides heterologous to the bacteria and / or the recipient, wherein the one or more polynucleotides encode one or more therapeutic polypeptides; and e) administering or causing the administration of at least a portion of the substantially homogeneous transformed population of isolated and cultured bacterial cells to a recipient subject, e.g., in a therapeutically sufficient amount, wherein the administered bacterial cells may permanently or long-term colonize or become colonized in or on the mammalian subject and express one or more therapeutic polypeptides, e.g., at a level sufficient to exert a therapeutic effect in the mammal. In some embodiments, the method further comprises determining and / or measuring the colonization or presence of the administered bacterial cells in or on the mammalian subject. In some embodiments, the microbiome sample is obtained from a biological sample selected from the group consisting of bodily excretions (e.g., stool, saliva, mucus, urine, breath), surface biopsies or swabs (e.g., gastrointestinal (GI) tract, oral cavity, pharynx, nasal cavity, urogenital tract, skin, anus / rectum, vagina, eye), and pathological specimens (e.g., cancerous tissue, amputated limb, inflamed organ). In some embodiments, the bacterial cells do not comprise polynucleotides encoding pathogenic toxins. In some embodiments, the bacterial cell or population of bacterial cells is a bacterial cell that is a member of the cytotoxin family, such as AB toxin, alpha toxin, anthrax toxin, botulinum toxin, cereulide, cholesterol-dependent cytolysin, Clostridial cytotoxin family, Clostridium botulinum C3 toxin, Clostridium difficile toxin, or the like. difficile toxin A, Clostridium difficile toxin B, Clostridial enterotoxins, Clostridium perfringens alpha toxin, Clostridium perfringens beta toxin, Cry1Ac, Cry6Aa, Cry34Ab1, delta endotoxin, diphtheria toxin, enterotoxin, enterotoxin type B, erythrogenic toxin, exfoliatin, fragilisin, hemolysin E, heat-labile enterotoxin, heat-stable enterotoxin, hemolysin, HrpZ family, leukocidin, listeriolysin O, Panton-Valentine leukocidin, intact pathogenicity island, phenol-soluble modulin, pneumolysin, pore-forming toxin, Pseudomonas exotoxin, pyocyanin, anti-eukaryotic Rhs toxin, RTX toxin, Shiga toxin,The bacterial cell or population of bacterial cells does not comprise one or more polynucleotides encoding one or more pathogenic toxins selected from the group consisting of Shiga-like toxin, Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin, Staphylococcus aureus delta toxin, streptolysin, tetanolysin, tetanospasmin, toxic shock syndrome toxin, tracheal cytotoxin, and / or Vero cytotoxin. In some embodiments, the bacterial cell or population of bacterial cells is antibiotic-sensitive to one or more antibiotic agents used to select transformed bacterial cells, such as kanamycin, chloramphenicol, carbenicillin, hygromycin, and / or trimethoprim. In some embodiments, the bacterial cell is not antibiotic-resistant to antibiotic agents used clinically. In some embodiments, the bacterial cell or population of bacterial cells is treated with a macrolide antibiotic (e.g., azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylocine, roxithromycin), a rifamycin (e.g., rifampicin (or rifampin), rifabutin, rifapentine, rifalazil, rifaximin), a polymyxin (e.g., polymyxin B, polymyxin E (colistin)), Quinolone antibiotics (e.g., nalidixic acid, ofloxacin, levofloxacin, ciprofloxacin, norfloxacin, enoxacin, lomefloxacin, grepafloxacin, trovafloxacin, sparfloxacin, temafloxacin, moxifloxacin, gatifloxacin, gemifloxacin), beta-lactams (e.g., penicillin, cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin), aminoglycosides (e.g., amikacin, gentamicin, neomycin,streptomycin, tobramycin), cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cefaclor, cefoxitin, cefprozil, cefuroxime, loracarbef, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, cefepime, ceftobiprole), monobactams (e.g., aztreonam, tigemonam, nocardicin A, tabtoxinin β-lactam), carbapenems (e.g., In some embodiments, the one or more heterologous polynucleotides encode a fluorescent protein, such as a green fluorescent protein, a yellow fluorescent protein, a red fluorescent protein (mCherry, mEos2, mRuby2, mRuby3, mClover3, mApple, mKate2, mMaple, mCardinal, or mNeptune), mTurquoise, or mVenus. In some embodiments, the one or more heterologous polynucleotides encode an enzyme, cytokine, or peptide hormone. In some embodiments, the enzyme is a bile salt hydrolase, such as a bile salt hydrolase from Lactobacillus, e.g., bshA (gene ID 3251811) or bshB (gene ID 3252955), N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D, dispersin B (DspB) from Actinobacillus actinomycetemcomitans, lactase (beta-galactosidase), aldehyde dehydrogenase, alcohol dehydrogenase (e.g., ADH1A, ADH1B, ADH1C, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7), bile acid-CoA:amino acid N-acyltransferase (BAAT),phenylalanine hydroxylase, butyrate synthesis pathway enzymes, prolyl endoprotease from Aspergillus niger (AN-PEP), 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH), 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), cholylglycine hydrolase, and cholic acid 7alpha-dehydroxylase. In some embodiments, the cytokine is selected from the group consisting of mammalian (e.g., human) IL-10 and mammalian (e.g., human) IL-27 dimer (IL27 alpha subunit and Epstein-Barr virus-induced 3 (EBI3) subunit expressed separately or as a fusion protein), and TGF-β. In some embodiments, the peptide hormone is selected from the group consisting of mammalian glucagon, glucagon-like peptide 1 (GLP-1), mammalian glucagon-like peptide 2 (GLP-2), fibroblast growth factor 1 (FGF1), fibroblast growth factor 15 (FGF15), fibroblast growth factor 19 (FGF19), insulin, and proinsulin. In some embodiments, the one or more heterologous polynucleotides encode Amuc_1100 from Akkermansia muciniphila, flagellin B from Vibrio vulnificus, elafin, trefoil factor 1 (TFF1), trefoil factor 2 (TFF2), trefoil factor 3 (TFF3), an anti-TNFα antibody / nanobody or fragment or single chain thereof, cyanovirin-N from Nostoc elipsosporum, or microcin J25 (MccJ25). In some embodiments, the one or more heterologous polynucleotides comprise a codon bias and / or codon optimization configured to improve or enhance expression of a heterologous protein in a transformed population of isolated and cultured bacterial cells. In some embodiments, the one or more heterologous polynucleotides are integrated into the chromosome of a bacterial cell of the transformed population. In some embodiments, the one or more heterologous polynucleotides are integrated into the attB and / or yfgG genes of the bacterial genome. In some embodiments,The one or more heterologous polynucleotides are in a plasmid that is episomally introduced into the bacterial cells of the transformed population. In some embodiments, the transformed bacterial cells further comprise a plasmid retention or maintenance system, such as a partitioning system or a toxin-antitoxin module or system. In some embodiments, the one or more heterologous polynucleotides are integrated into an expression cassette having at least or at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:2 and are expressed under the control of a Ptrc promoter. In some embodiments, the heterologous polynucleotide is expressed under the control of a constitutive promoter. In some embodiments, the heterologous polynucleotide is expressed under the control of an inducible promoter. In some embodiments, the bacterial cells are derived from a Gram-negative bacterial strain. In some embodiments, the bacterial cell is from a bacterial genus selected from the group consisting of Bacteroides (e.g., Alistipes, Prevotella, Paraprevotella, Parabacteroides, or Odoribacter), Clostridium, Streptococcus, Lactococcus, Eubacterium rectale, Escherichia coli, Enterobacter species, Klebsiella species, Bifidobacterium, Staphylococcus, Lactobacillus, Veillonella, Haemophilus, Moraxella, Corynebacterium, and Propionibacterium. In some embodiments, the bacterial cell is from E. coli. In some embodiments, the detectable portion of the administered bacterial cells stably resides in the tissue or surface to which it is administered for at least or at least about 2, 3, 4, 5, 6, 7 days, e.g., at least or at least about 1 week, e.g., at least or at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 125 weeks, or longer, e.g., for the duration of the subject's life, or for a period within a range defined by any two of the aforementioned periods. In some embodiments, the detectable portion of the administered bacterial cells stably resides permanently in the tissue or surface to which it is administered. In some embodiments,At least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the administered bacterial cells stably colonize the tissue or surface to which they are administered. In some embodiments, the native / symbiotic host cells (i) are capable of metabolizing one or more carbohydrates selected from the group consisting of sucrose, xylose, d-maltose, N-acetyl-d-glucosamine, d-galactose, and d-ribose; (ii) utilize both glycolytic and gluconeogenic substrates; (iii) are non-motile (e.g., have a non-functional flagellum, e.g., due to a mutation in the flhDC operon); (iv) are capable of producing ribose-5-phosphate; and (v) grow in a defined medium lacking vitamin B12 (cyanocobalamin). (vi) expressing UDP-glucose-4-epimerase and / or glycosyltransferase; (vii) containing multiple copies of the gene encoding the β subunit of the tryptophan synthase gene; (viii) containing multiple copies of the gene encoding propionate CoA-transferase; (ix) expressing capsular polysaccharide (CPS) 4 (CPS4); (x) expressing an rnf-like oxidoreductase complex; (xi) catabolizing tryptophan to produce indole and and other indole metabolites, e.g., indole-3-propionate and indole-3-aldehyde; and / or do not produce any substances that induce double-stranded DNA breaks, e.g., do not have genomic islands encoding large modular non-ribosomal peptide and polyketide synthases, do not express hybrid peptide-polyketide genotoxic substances, and / or do not have an active clbA gene. In some embodiments, the subject is a human. In some embodiments, the subject is at least or at least about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 1013 In some embodiments, the donor subject and recipient subject are the same individual, e.g., the microbiome sample is autologous to the subject. In some embodiments, the donor subject and recipient subject are different individuals. In some embodiments, the microbiome sample is from a mammal of the same species as the subject. In some embodiments, the administered bacterial cells are administered to the same tissue or surface from which the microbiome sample was obtained. In some embodiments, the microbiome sample is obtained from the skin or eye, and the population of bacterial cells is administered to the subject topically, e.g., in the form of a buffered suspension, gel, lotion, cream, or ointment. In some embodiments, the microbiome sample is obtained from the nasal cavity, and the administered bacterial cells are administered via nasal gavage. In some embodiments, the microbiome sample is obtained from the vagina, and the administered bacterial cells are administered intravaginally. In some embodiments, the microbiome sample is obtained from the gastrointestinal tract, and the administered bacterial cells are administered orally or rectally to the subject. In some embodiments, the administered bacterial cells are administered to the subject orally via a gastric tube or in the form of an edible composition. In some embodiments, the edible composition comprises a gel capsule containing the administered bacterial cells, or the administered bacterial cells are encapsulated. In some embodiments, the edible composition is selected from the group consisting of yogurt, milk, ice cream, vegetable puree, fruit puree, sherbet, and oatmeal. In some embodiments, the edible composition is a beverage. In some embodiments, the beverage is a buffered solution. In some embodiments, the administered bacterial cells are administered to the subject multiple times, for example, at daily, weekly, biweekly, or monthly intervals. In some embodiments, the administered bacterial cells are administered to the subject at daily, weekly, biweekly, or monthly intervals. In some embodiments, administration of the transformed bacterial cells does not alter the recipient subject's microbiome.

[0007] In a further aspect, a substantially homogeneous population of bacterial cells symbiotic to a mammal is provided, wherein the population of bacteria is transformed to express one or more polynucleotides heterologous to the mammal and / or the bacteria. In some embodiments, the population of bacteria native to or symbiotic to the mammal is capable of stably, permanently, or long-term establishment in or on the mammal, e.g., for at least or at least about 2, 3, 4, 5, 6, 7 days, e.g., at least or at least about 1 week, e.g., at least or at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 125 weeks, or longer, e.g., for the duration of the mammal's life. In some embodiments, the native / symbiotic host cell (i) is capable of metabolizing one or more carbohydrates selected from the group consisting of sucrose, xylose, d-maltose, N-acetyl-d-glucosamine, d-galactose, and d-ribose; (ii) utilizes both glycolytic and gluconeogenic substrates; (iii) is non-motile (e.g., has a non-functional flagellum, e.g., due to a mutation in the flhDC operon); (iv) is capable of producing ribose-5-phosphate; (v) is capable of growth in defined medium lacking vitamin B12 (cyanocobalamin) (e.g., vitamin B12 prototrophy has been demonstrated); (vi) exhibits UDP-glucose-4-epimerase and / or glycosyltransferase. (vii) contain multiple copies of the gene encoding the β subunit of the tryptophan synthase gene; (viii) contain multiple copies of the gene encoding propionate CoA-transferase; (ix) express capsular polysaccharide (CPS) 4 (CPS4); (x) express an rnf-like oxidoreductase complex; (xi) catabolize tryptophan to produce indole and other indole metabolites, such as indole-3-propionate and indole-3-aldehyde; and / or do not produce any substances that induce double-stranded DNA breaks, e.g., do not have genomic islands encoding large modular non-ribosomal peptide and polyketide synthases.In some embodiments, the population of bacterial cells does not express a hybrid peptide-polyketide genotoxic agent and / or does not have an active clbA gene. In some embodiments, the population of bacterial cells expresses or is expressing any of the following: AB toxin, alpha toxin, anthrax toxin, botulinum toxin, cereulide, cholesterol-dependent cytolysin, Clostridial cytotoxin family, Clostridium botulinum C3 toxin, Clostridium difficile toxin A, Clostridium difficile toxin B, Clostridial enterotoxin, Clostridium perfringens alpha toxin, Clostridium perfringens beta toxin, Cry1Ac, Cry6Aa, Cry34Ab1, delta endotoxin, diphtheria toxin, enterotoxin, enterotoxin type B, erythrogenic toxin, exfoliatin, fragilisin, hemolysin E, heat-labile enterotoxin, heat-stable enterotoxin, The bacterial cell population does not comprise one or more polynucleotides encoding one or more pathogenic toxins selected from the group consisting of arotoxin, hemolysin, HrpZ family, leukocidin, listeriolysin O, Panton-Valentine leukocidin, intact pathogenicity island, phenol-soluble modulin, pneumolysin, pore-forming toxin, Pseudomonas exotoxin, pyocyanin, anti-eukaryotic Rhs toxin, RTX toxin, Shiga toxin, Shiga-like toxin, Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin, Staphylococcus aureus delta toxin, streptolysin, tetanolysin, tetanospasmin, toxic shock syndrome toxin, tracheal cytotoxin, and / or Vero cytotoxin. In some embodiments, the population of bacterial cells is antibiotic-resistant to one or more antibiotic agents used to select for transformed bacterial cells, e.g., kanamycin, chloramphenicol, carbenicillin, hygromycin, and / or trimethoprim. In some embodiments, the bacterial cells are not antibiotic-resistant to clinically used antibiotic agents. In some embodiments, the bacterial cells are not antibiotic-resistant to macrolide antibiotics (e.g., azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin,spiramycin, troleandomycin, tylosin / tylocine, roxithromycin), rifamycins (e.g., rifampicin (or rifampin), rifabutin, rifapentine, rifalazil, rifaximin), polymyxins (e.g., polymyxin B, polymyxin E (colistin)), quinolone antibiotics (e.g., nalidixic acid, ofloxacin, levofloxacin, ciprofloxacin, norfloxacin, enoxacin, lomefloxacin) flucloxacin, grepafloxacin, trovafloxacin, sparfloxacin, temafloxacin, moxifloxacin, gatifloxacin, gemifloxacin), beta-lactams (e.g., penicillin, cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin), aminoglycosides (e.g., amikacin, gentamicin, isin, neomycin, streptomycin, tobramycin), cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cefaclor, cefoxitin, cefprozil, cefuroxime, loracarbef, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, cefepime, ceftobiprole), monobactams (e.g., aztreonam, tigemonam, nocardicin A, tabtoxinin β-lactam), carbapenems (e.g., In some embodiments, the one or more heterologous polynucleotides are not antibiotic-resistant to one or more clinically used antibiotic agents selected from, for example, biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, thienamycin), and tetracyclines (e.g., tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, tigecycline). In some embodiments, the one or more heterologous polynucleotides are selected from the group consisting of fluorescent proteins, e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein (mCherry, mEos2, mRuby2, mRuby3, mClover3, mApple,In some embodiments, the one or more heterologous polynucleotides encode an enzyme, cytokine, or peptide hormone. In some embodiments, the enzyme is a bile salt hydrolase, e.g., a bile salt hydrolase from Lactobacillus genus, e.g., bshA (gene ID 3251811) or bshB (gene ID 3252955), N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D, dispersin B of Actinobacillus actinomycetemcomitans (DspB), lactase (beta-galactosidase), aldehyde dehydrogenase, alcohol dehydrogenase (e.g., ADH1A, ADH1B, ADH1C, AD In some embodiments, the cytokine is selected from the group consisting of mammalian (e.g., human) IL-10 and mammalian (e.g., human) IL-27 dimer (IL-27 alpha subunit and Epstein-Barr virus-induced 3 (EBI3) subunit expressed separately or as a fusion protein), and TGF-β. In some embodiments, the peptide hormone is selected from the group consisting of mammalian glucagon, glucagon-like peptide 1 (GLP-1), mammalian glucagon-like peptide 2 (GLP-2), fibroblast growth factor 1 (FGF1), fibroblast growth factor 15 (FGF15), fibroblast growth factor 19 (FGF19), insulin, and proinsulin. In some embodiments, the one or more heterologous polynucleotides are selected from the group consisting of Akkermansia muciniphila Amuc_1100, Vibrio vulnificus flagellin B, elafin, trefoil factor 1 (TFF1),In some embodiments, the one or more heterologous polynucleotides encode trefoil factor 2 (TFF2), trefoil factor 3 (TFF3), an anti-TNFα antibody / nanobody or fragment or single chain thereof, Nostoc ellipsosporum cyanovirin-N, or microcin J25 (MccJ25). In some embodiments, the one or more heterologous polynucleotides comprise codon bias and / or codon optimization configured to improve or enhance expression of the heterologous protein in the transformed population of isolated and cultured bacterial cells. In some embodiments, the one or more heterologous polynucleotides are integrated into the chromosome of the bacterial cells of the transformed population. In some embodiments, the one or more heterologous polynucleotides are integrated into the attB and / or yfgG genes of the bacterial genome. In some embodiments, the heterologous polynucleotides are in a plasmid episomally disposed in the bacterial cell. In some embodiments, the transformed bacterial cell further comprises a plasmid maintenance or maintenance system, such as a partitioning system or a toxin-antitoxin module or system. In some embodiments, the one or more heterologous polynucleotides are integrated into an expression cassette having at least or at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 2 and are expressed under the control of the Ptrc promoter. In some embodiments, the substantially homogenous population of bacterial cells is derived from a Gram-negative bacterial strain. In some embodiments, the substantially homogenous population of bacterial cells is from a bacterial genus selected from the group consisting of Bacteroides (e.g., Alistipes, Prevotella, Paraprevotella, Parabacteroides, or Odoribacter), Clostridium, Streptococcus, Lactococcus, Eubacterium rectare, Escherichia coli, Enterobacter species, Klebsiella species, Bifidobacterium, Staphylococcus, Lactobacillus, Veillonella, Haemophilus, Moraxella, Corynebacterium, and Propionibacterium. In some embodiments, the substantially homogenous population of bacterial cells is from Escherichia coli. In some embodiments, the population of bacterial cells is lyophilized or cryopreserved.

[0008] In another aspect, pharmaceutical compositions suitable for administration to a mammal, e.g., suitable for delivery of one or more therapeutic polypeptides to a mammal, are provided. In another aspect, edible compositions are provided. In some embodiments, the composition comprises a substantially homogeneous population of bacterial cells that are symbiotic to the mammal, as described above and herein, wherein the population of bacteria is transformed to express one or more polynucleotides that are heterologous to the mammal and / or the bacteria. In some embodiments, the population of bacteria that are native to or symbiotic to the mammal is produced according to the methods described above and herein. In some embodiments, the population of bacteria that are symbiotic to a mammal is capable of or configured to establish in or on the mammal permanently or for an extended period of time, e.g., for at least or at least about 2, 3, 4, 5, 6, 7 days, e.g., at least or at least about 1 week, e.g., at least or at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 125 weeks, or more, e.g., for the duration of the mammal's life. In some embodiments, the native / symbiotic host cells (i) are capable of metabolizing one or more carbohydrates selected from the group consisting of sucrose, xylose, d-maltose, N-acetyl-d-glucosamine, d-galactose, and d-ribose; (ii) utilize both glycolytic and gluconeogenic substrates; (iii) are non-motile (e.g., have non-functional flagella,(iv) are capable of producing ribose-5-phosphate; (v) are capable of growing in defined media lacking vitamin B12 (cyanocobalamin) (e.g., vitamin B12 prototrophy has been demonstrated); (vi) express UDP-glucose-4-epimerase and / or glycosyltransferase; (vii) contain multiple copies of the gene encoding the β subunit of the tryptophan synthase gene; (viii) contain multiple copies of the gene encoding propionate CoA-transferase; (ix) ) express capsular polysaccharide (CPS) 4 (CPS4); (x) express an rnf-like oxidoreductase complex; (xi) catabolize tryptophan to produce indole and other indole metabolites, e.g., indole-3-propionate and indole-3-aldehyde; and / or do not produce any substances that induce double-stranded DNA breaks, e.g., do not have genomic islands encoding large modular non-ribosomal peptide and polyketide synthases, do not express hybrid peptide-polyketide genotoxic substances, and / or do not have an active clbA gene. In some embodiments, the population of bacterial cells is a bacterial cell that is a member of the cytotoxin family, such as AB toxin, alpha toxin, anthrax toxin, botulinum toxin, cereulide, cholesterol-dependent cytolysin, Clostridial cytotoxin family, Clostridium botulinum C3 toxin, Clostridium difficile toxin A, Clostridium difficile toxin B, Clostridial enterotoxin, Clostridium perfringens alpha toxin, Clostridium perfringens beta toxin, Cry1Ac, Cry6Aa, Cry34Ab 1, delta-endotoxin, diphtheria toxin, enterotoxin, enterotoxin type B, erythrogenic toxin, exfoliatin, fragilisin, hemolysin E, heat-labile enterotoxin, heat-stable enterotoxin, hemolysin, HrpZ family, leukocidin, listeriolysin O, Panton-Valentine leukocidin, intact pathogenicity island, phenol-soluble modulin, pneumolysin, pore-forming toxin, Pseudomonas exotoxin, pyocyanin, anti-eukaryotic Rhs toxin, RTX toxin, Shiga toxin, Shiga-like toxin,The bacterial cell population does not comprise one or more polynucleotides encoding one or more pathogenic toxins selected from the group consisting of Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin, Staphylococcus aureus delta toxin, streptolysin, tetanolysin, tetanospasmin, toxic shock syndrome toxin, tracheal cytotoxin, and / or Vero cytotoxin. In some embodiments, the population of bacterial cells is antibiotic-resistant to one or more antibiotic agents used to select the transformed bacterial cells, such as kanamycin, chloramphenicol, carbenicillin, hygromycin, and / or trimethoprim. In some embodiments, the bacterial cell or population of bacterial cells is not antibiotic-resistant to antibiotic agents used clinically. In some embodiments, the bacterial cell or population of bacterial cells is / are treated with a macrolide antibiotic (e.g., azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylocine, roxithromycin), a rifamycin (e.g., rifampicin (or rifampin), rifabutin, rifapentine, rifalazil, rifaximin), a polymyxin (e.g., polymyxin B, polymyxin E (colistin)), a quinolone antibiotic (e.g., nalidixic acid, ofloxacin, sparfloxacin, levofloxacin, ciprofloxacin, norfloxacin, enoxacin, lomefloxacin, grepafloxacin, trovafloxacin, sparfloxacin, temafloxacin, moxifloxacin, gatifloxacin, gemifloxacin), beta-lactams (e.g., penicillin, cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin), aminoglycosides (e.g., amikacin, gentamicin, neomycin, streptomycin, tobramycin), cephalosporins (e.g., cefadroxil, cefazolin,Cephalexin, cefaclor, cefoxitin, cefprozil, cefuroxime, loracarbef, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, cefepime, ceftobiprole), monobactams (e.g., aztreonam, tigemonam, nocardicin A, tabtoxin β-lactam), carbapenems (e.g., biapenem, doripenem, ertapenem, faropenem, imipenem, In some embodiments, the one or more heterologous polynucleotides encode a fluorescent protein, such as a green fluorescent protein, a yellow fluorescent protein, a red fluorescent protein (mCherry, mEos2, mRuby2, mRuby3, mClover3, mApple, mKate2, mMaple, mCardinal, or mNeptune), mTurquoise, or mVenus. In some embodiments, the one or more heterologous polynucleotides encode an enzyme, a cytokine, or a peptide hormone. In some embodiments, the enzyme is a bile salt hydrolase, e.g., a bile salt hydrolase from Lactobacillus sp., e.g., bshA (gene ID 3251811) or bshB (gene ID 3252955), N-acylphosphatidylethanolamine (NAPE) hydrolyzing phospholipase D, dispersin B (DspB) from Actinobacillus actinomycetemcomitans, lactase (beta-galactosidase), aldehyde dehydrogenase, alcohol dehydrogenase (e.g., ADH1A, ADH1B, ADH1C, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7), bile acid-CoA:amino acid N-acyltransferase (BAAT), phenylalanine hydroxylase, butyrate synthesis pathway enzyme, prolyl endoprotease from Aspergillus niger (AN-PEP),7alpha-hydroxysteroid dehydrogenase (7-alpha-HSDH), 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), cholylglycine hydrolase, and cholic acid 7alpha-dehydroxylase. In some embodiments, the cytokine is selected from the group consisting of mammalian (e.g., human) IL-10 and mammalian (e.g., human) IL-27 dimer (IL27 alpha subunit and Epstein-Barr virus-induced 3 (EBI3) subunit expressed separately or as a fusion protein), and TGF-β. In some embodiments, the peptide hormone is selected from the group consisting of glucagon, mammalian glucagon-like peptide 1 (GLP-1), mammalian glucagon-like peptide 2 (GLP-2), fibroblast growth factor 1 (FGF1), fibroblast growth factor 15 (FGF15), fibroblast growth factor 19 (FGF19), insulin, and proinsulin. In some embodiments, the one or more heterologous polynucleotides encode Amuc_1100 from Akkermansia muciniphila, flagellin B from Vibrio vulnificus, elafin, trefoil factor 1 (TFF1), trefoil factor 2 (TFF2), trefoil factor 3 (TFF3), an anti-TNFα antibody / nanobody or fragment or single chain thereof, cyanovirin-N from Nostoc ellipsosporum, or microcin J25 (MccJ25). In some embodiments, the one or more heterologous polynucleotides comprise codon bias and / or codon optimization configured to improve or enhance expression of a heterologous protein in a transformed population of isolated and cultured bacterial cells. In some embodiments, the one or more heterologous polynucleotides are integrated into the chromosome of a bacterial cell of the transformed population. In some embodiments, the one or more heterologous polynucleotides are integrated into the attB and / or yfgG genes of the bacterial genome. In some embodiments, the heterologous polynucleotide is in a plasmid that is episomally located in the bacterial cell. In some embodiments, the transformed bacterial cell further comprises a plasmid retention or maintenance system, such as a partitioning system or a toxin-antitoxin module or system. In some embodiments,The one or more heterologous polynucleotides are integrated into an expression cassette having at least or at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 2 and are expressed under the control of the Ptrc promoter. In some embodiments, the substantially homogenous population of bacterial cells is derived from a Gram-negative bacterial strain. In some embodiments, the substantially homogenous population of bacterial cells is from a bacterial genus selected from the group consisting of Bacteroides (e.g., Alistipes, Prevotella, Paraprevotella, Parabacteroides, or Odoribacter), Clostridium, Streptococcus, Lactococcus, Eubacterium rectare, Escherichia coli, Enterobacter species, Klebsiella species, Bifidobacterium, Staphylococcus, Lactobacillus, Veillonella, Haemophilus, Moraxella, Corynebacterium, and Propionibacterium. In some embodiments, the substantially homogenous population of bacterial cells is from Escherichia coli. In some embodiments, the composition comprises a buffered solution or suspension. In some embodiments, the edible composition comprises a gel capsule comprising the administered bacterial cells, or the administered bacterial cells are encapsulated. In some embodiments, the edible composition comprises a beverage. In some embodiments, the edible composition is selected from the group consisting of yogurt, milk, ice cream, vegetable puree, fruit puree, sherbet, and oatmeal.

[0009] In a further aspect, a kit is provided that comprises one or more containers containing one or more compositions as described above and herein. In some embodiments, the population of bacterial cells is lyophilized.

[0010] definition The terms "commensal bacteria" or "native bacteria" refer synonymously to bacterial cells or populations of cells derived from, adapted to, or configured for colonization of a mammalian microbiome. Commensal bacteria are adapted to colonize or are configured for colonization of a mammal (e.g., bodily excretions (e.g., saliva, mucus, urine, or stool) or surfaces (e.g., mucosal gastrointestinal tract, mouth / pharynx / nares, urogenital tract, skin, anus / rectum, cheek / mouth, or eyes) and are not adapted or configured for cultivation in a laboratory environment.

[0011] "Administering," as used herein, refers to local and systemic administration, such as enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration for engineered natural bacteria (ENB) that find use in the methods described herein include, for example, oral (per os (PO)), rectal (e.g., administration as a suppository), vaginal, nasal, or inhalation, topical contact (e.g., to the skin or eye), or intralesional administration to a subject. Administration can be by any route, including parenteral and / or transmucosal (e.g., oral, nasal, vaginal, or rectal). Administration can be performed by a healthcare professional or can include self-administration.

[0012] The terms "systemic administration" and "administered systemically" refer to a method of administering a compound or composition to a mammal such that the compound or composition is delivered via the circulatory system to a body site, such as a targeted site of pharmaceutical action. Systemic administration includes, but is not limited to, oral, nasal, and / or rectal administration.

[0013] The phrase "administered" refers to an action taken by a medical professional (e.g., a physician) or a person managing a subject's medical care who administers and / or authorizes the administration of the agent / compound in question to a subject. Administering may include diagnosing and / or determining an appropriate therapeutic or prophylactic regimen for a subject, and / or prescribing a particular agent / compound. Such prescribing may include, for example, drafting a prescription form, annotating a medical record, etc.

[0014] The term "co-administering" or "simultaneous administration" refers to administering multiple ENB populations or one or more ENB populations and another active agent so that both achieve a physiological effect simultaneously. However, the two agents do not necessarily need to be administered together. In certain embodiments, administration of one agent can occur before administration of the other. A simultaneous physiological effect does not necessarily require both agents to be present in the circulation at the same time. However, in certain embodiments, co-administration typically results in a situation in which both agents, at any given dose, are simultaneously present in the body (e.g., in plasma) at a significant fraction (e.g., 20% or more, preferably 30% or 40% or more, more preferably 50% or 60% or more, and most preferably 70%, 80%, or 90% or more) of their peak serum concentrations.

[0015] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage of one or more compounds, and / or dosing regimen necessary to bring about a desired result, e.g., an amount sufficient to alleviate, in a mammal, one or more symptoms associated with the disease state for which the subject is receiving therapy, or an amount sufficient to lessen the severity or delay the progression of the disease state in a mammal (e.g., a therapeutically effective amount), an amount sufficient to reduce the risk or delay the onset and / or reduce the ultimate severity of the disease state in a mammal (e.g., a prophylactically effective amount).

[0016] The terms "treat" and "treatment," as used herein, refer to delaying the onset of, slowing or reversing the progression of, reducing the severity of, or alleviating or preventing any of the diseases or conditions to which the terms apply, or one or more symptoms of such diseases or conditions.

[0017] The term "alleviating" refers to the reduction or elimination of one or more symptoms of the pathology or disease and / or a reduction in the rate of, or delay in the onset or severity of, one or more symptoms of the pathology or disease and / or prevention of the pathology or disease.

[0018] The terms "subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to domestic mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and / or agricultural mammals (e.g., horses, cows, pigs, or sheep). In various embodiments, a subject may be a human (e.g., an adult male, adult female, juvenile male, juvenile female, boy, or girl) under the care of a physician or other medical professional in a hospital, psychiatric facility, as an outpatient, or in other clinical settings. In certain embodiments, a subject may not be receiving the care or prescription of a physician or other medical professional.

[0019] A "substantially homogenous population of bacterial cells," as used herein, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% genetically identical as determined, for example, by whole genome sequencing or by sequencing of ribosomal RNA 16S DNA.

[0020] As used herein, an engineered native bacterial (ENB) cell "stably establishes" means that it establishes and divides (e.g., reproduces) itself in or near the lumen or tissue to which it is administered, e.g., for at least 3, 4, 5, or 6 days, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 125 weeks, or longer, e.g., for the duration of the subject's life, or for a period within a range defined by any two of the aforementioned periods.

[0021] The term "heterologous nucleic acid" or "heterologous polypeptide" refers to a nucleic acid or polypeptide whose sequence is not identical to the sequence of another nucleic acid or polypeptide that is naturally found in the same host cell or host. A "heterologous nucleic acid" or "heterologous polypeptide," as used herein, may be heterologous to a bacterial cell and / or a mammalian host.

[0022] The terms "transform" or "transformation," as used herein, refer to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically stable inheritance. Host bacterial cells containing the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.

[0023] The term "therapeutic polypeptide" refers to a polypeptide that has therapeutic pharmacological activity in a mammal.

[0024] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same, or have a specified percentage of amino acid residues or nucleotides that are the same, i.e., have at least or at least about 80% identity, e.g., at least or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, over a specified region, to a reference sequence, e.g., a heterologous polynucleotide or polypeptide sequence listed in Table 1, when compared and aligned for maximum correspondence over a comparison window, or over an indicated region, as measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. Such sequences are then referred to as "substantially identical." This definition also refers to the complementarity of a test sequence. Identity preferably exists over a region that is at least or at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0025] For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. BLAST and BLAST 2.0 algorithms and default parameters are used to compare nucleic acid and protein sequences with reference nucleic acids and proteins.

[0026] Two nucleic acid sequences or polypeptides are said to be substantially identical if the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another way to say that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another way to say that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences. [Brief explanation of the drawings]

[0027] [Figure 1]

[0023] Figure 1 illustrates a schematic diagram of the methods described herein. Naturally occurring (i.e., commensal) bacteria can be used as vectors to introduce new functions (e.g., delivery of therapeutic polypeptides, e.g., bile salt hydrolase) into the microbiome (e.g., gut, skin) of a normally developing wild-type host (e.g., human). [Figure 2]This figure illustrates a schematic diagram of how engineered bacteria expressing BSH can affect neuroinflammation and cognition. Bile acids (BAs) are key mediators of the microbiome-gut-brain axis. Luminal BA deconjugation affects neuroinflammation and cognitive performance in diet-induced obese mice. [Figure 3A] 1 shows the action of bile salt hydrolase (BSH) on taurocholate. BSH deconjugates bile acids, converting, for example, taurocholate (TCA) to cholic acid (CA). This action makes the bile acid more difficult to reabsorb, allowing it to be further processed by other bacteria in the gut microbiome. [Figure 3B] FIG. 1 shows that native E. coli (here identified as ENB) can be genetically modified to express BSH, as demonstrated by the deconjugation of TCA to CA in vitro with cells in PBS buffer. [Figure 3C] Native E. coli (top left) can be engineered to express GFP (top right) and further engineered to express bile salt hydrolase (bottom) that can deconjugate TDCA (soluble) to DCA (insoluble—white halo). [Figure 4A] Colonization of mice with genetically engineered derivatives of strain AZ-39. After a single gavage (n=12-16 up to 100 days; n=3-8 for an additional 300 days) under different dietary conditions (e.g., feeding both normal solid food and high-fat diets), engineered and native E. coli remain at stable levels for more than a year. [Figure 4B] Engineered and native bacteria colonize the entire gastrointestinal tract, particularly the distal gastrointestinal tract. Engineered and native E. coli colonize the entire gastrointestinal tract, with the majority concentrated in the terminal ileum and cecum (n=4-6). [Figure 4C]This figure shows that engineered native bacteria do not affect normal weight gain or diet-induced obesity in mice. Colonization of mice with engineered native E. coli did not affect body weight in both high-fat and normal chow diets (n = 12-16). Note that previous reports on the expression of this gene in immune-reduced and germ-free mice suggested that BSH would be expected to affect adiposity, but not in normally developing wild-type mice. [Figure 4D-E] Figure 4F: Colonization of germ-free mice with engineered native bacteria expressing GFP did not affect fecal bile acid composition. However, engineered native bacteria expressing both GFP and BSH showed significantly higher levels of deconjugated bile acids (n=3). [Figure 4F] Engineered and native E. coli expressing BSH deconjugate bile acids in germ-free mice (blue: uncolonized mice; green: mice colonized with AZ-39 expressing GFP; red: mice colonized with AZ-39 expressing GFP and BSH). Engineered and native bacteria expressing both GFP and BSH (red) showed significantly higher levels of deconjugated bile acids, especially TCA and TbMCA (n=3). There were no significant differences in other unconjugated / conjugated bile acids. [Figure 5-1]Figures 5A-C: Engineered native bacteria do not alter the composition of the gut microbiome. The addition of recombinant engineered native bacteria (with or without BSH) does not alter the gut microbiome in a way detectable by 16S sequencing and analysis 10 weeks after colonization (top panel shows weighted PCoA, unweighted PCoA, and Bray-Curtis distances). However, in correlation analysis, specific OTUs related to the genus Bacteroides became highly correlated with BSH-expressing E. coli, suggesting a shift in the relationships between bacteria in the gut, even if the overall composition remains unchanged. Figures 5B-C: Engineered native bacteria affect both fecal and serum bile acid pools. Fecal (top) and serum (bottom) bile acid correlograms. The addition of native bacteria with bile salt hydrolase caused only minor changes in fecal bile acids (see, for example, Figure 6). However, native bacteria with BSH caused significant changes in serum bile acids. In these mice, serum BAs showed an inverse correlation between conjugated and deconjugated BAs. [Figure 5-2]Figures 5A-C: Engineered native bacteria do not alter the composition of the gut microbiome. The addition of recombinant engineered native bacteria (with or without BSH) does not alter the gut microbiome in a way detectable by 16S sequencing and analysis 10 weeks after colonization (top panel shows weighted PCoA, unweighted PCoA, and Bray-Curtis distances). However, in correlation analysis, specific OTUs related to the genus Bacteroides became highly correlated with BSH-expressing E. coli, suggesting a shift in the relationships between bacteria in the gut, even if the overall composition remains unchanged. Figures 5B-C: Engineered native bacteria affect both fecal and serum bile acid pools. Fecal (top) and serum (bottom) bile acid correlograms. The addition of native bacteria with bile salt hydrolase caused only minor changes in fecal bile acids (see, for example, Figure 6). However, native bacteria with BSH caused significant changes in serum bile acids. In these mice, serum BAs showed an inverse correlation between conjugated and deconjugated BAs. [Figure 6A] Figure 1 shows that engineered native bacteria affect fecal bile acids. Examples of fecal bile acids affected by our engineered native bacteria. In mice receiving native bacteria engineered to express BSH, tauro-beta-muricholic acid (TbMCA; left) and TCA (right) were significantly lower. In these mice, TDCA (center) was significantly higher. DCA is a secondary bile acid, and microbial synthesis of DCA requires deconjugation as a first step. [Figure 6B] Figure 1 shows that engineered native bacteria affect serum bile acids. There was a more dramatic difference in serum bile acids than in fecal bile acids. In mice receiving engineered bacteria expressing BSH, levels of beta-muricholic acid (bMCA), tauro-bMCA, and omega-muricholic acid (oMCA) were reduced. [Figure 7A]Figure 1 shows that engineered native bacteria (ENB) expressing BSH alter global metabolism: Measurement of respiratory quotient (VCO2 / VO2; RER) in engineered bacteria expressing BSH and those not expressing BSH. Mice colonized with engineered native bacteria expressing BSH had a significantly lower RER than mice colonized with engineered native bacteria not expressing BSH. This suggests that these mice preferentially use fatty acids over carbohydrates for their metabolism. [Figure 7B] Engineered native bacteria can alter host physiology. Mice receiving native bacteria expressing BSH had normal fasting insulin levels (left). However, their postprandial insulin levels (right) were significantly lower than those of the control cohort, suggesting greater insulin sensitivity. [Figure 7C] Figure 1 shows that engineered native bacteria expressing BSH affect behavior: Mice containing engineered native bacteria expressing BSH (red) exercised approximately 50% more than mice without the bacteria (green) or mice with bacteria that do not contain the BSH gene (blue). [Figure 7D] Figure 1 shows that engineered native bacteria expressing BSH affect cognition. In a novel object recognition test, all mice on a normal chow diet spent more time on the novel object, regardless of whether they were colonized with engineered bacteria. However, as seen in previous studies, mice maintained on a high-fat diet were unable to distinguish between novel and old objects, suggesting that they had memory problems. However, mice receiving engineered native bacteria expressing BSH appeared to show a normal increase in interest in the novel object, thus restoring memory function. [Figure 8] Figure 1 illustrates that by administering naturally occurring bacteria engineered to express BSH, the inventors affected host physiology: Mice receiving engineered naturally occurring bacteria expressing BSH had lower fasting glucagon levels. [Figure 9A] 1 shows an oral glucose tolerance test in wild-type, normally developing, normal chow-fed mice colonized with engineered native bacteria. Engineered native bacteria expressing BSH resulted in lower postprandial insulin, but there was no difference in serum glucose levels whether the bacteria expressed BSH or not. [Figure 9B] Oral glucose tolerance test in normally developing ob / ob mice on a normal chow diet colonized with engineered native bacteria (blue: AZ51 / BSH-, orange: AZ52 / BSH+). Expression of BSH by engineered bacteria significantly improved insulin sensitivity in this animal model of obesity / type 2 diabetes. [Figure 10] Figure 1 illustrates the colonization of mice with engineered native bacteria regardless of food after a single gavage. NFD: no fat diet, VLFD: very low fat diet, LFD: low fat diet. [Figure 11] FIG. 1 illustrates that engineered native bacteria retain BSH activity 22 weeks after gavage. [Figure 12] Figure 1 illustrates retention of GFP and BSH gene expression after X weeks of gavage. Only a single isolate expected to have BSH activity did not (bold). [Figure 13] Figure 1 illustrates the antibiotic susceptibility of engineered and native E. coli strains. These strains do not contain genes homologous to beta-lactams. Simultaneous resistance to cephalexin and sensitivity to carbenicillin suggests specific resistance due to PBP mutations. [Figure 14] Figure 1 illustrates total bile acids in fecal pellets from colonized mice. The presence of the BSH gene (AZ-52) results in significantly greater loss of bile acids in the feces than the absence of the BSH gene (AZ-51; p<0.003). [Figure 15]Figure 1 illustrates that the chromosomal yfgG site for transgene integration is retained in 208 of 210 full-length E. coli genomes (NCBI GenBank nt database). A solid line indicates an intact insertion site; a vertically offset dashed line indicates a chromosomal rearrangement at that site. [Figure 16] Figure 1 illustrates that bacterial overgrowth is not associated with colonization, regardless of the presence or absence of BSH, 6 months after colonization. Terminal ileal tissue was flash-frozen, powdered, and total DNA extracted. Bacterial 16S copy number and host GAPDH copy number were assessed by quantitative PCR, and 16S abundance was normalized by host GAPDH copy number. [Figure 17] FIG. 1 illustrates that engineered native bacteria do not significantly alter the terminal ileum microbiome, regardless of the presence or absence of BSH. [Figure 18] Figure 1 illustrates that engineered native E. coli do not alter host fecal production 2 months after colonization. Colonized mice were housed singly and feces were collected every 3 hours for 48 hours. DETAILED DESCRIPTION OF THE INVENTION

[0028] 1. Introduction Few tools are available that allow researchers to functionally manipulate the gut microbiome and achieve a better mechanistic understanding of host-microbe relationships. We have developed a technology to "knock in" functions into the gut microbiome to investigate their effects on luminal ecology, metabolite and nutrient flow, and ultimately physiology in normally developing wild-type (CR-WT) mice (as opposed to mice raised in a germ-free environment, for example). We can achieve this by identifying and engineering tractable native bacteria (as opposed to laboratory strains or so-called commensal bacteria) to express genes of interest in the luminal environment.

[0029] The methods and compositions described herein circumvent the problems of traditional probiotic microorganisms by reinventing commensal organisms to provide therapeutic functionality. Commensal strains of microorganisms, by their very definition, are reservoirs of organisms capable of stable, long-term, or persistent colonization of at least one specific mammalian host. Current probiotics are single-strain organisms that, while used in multiple hosts, have not been very successful in broad populations.

[0030] Using known probiotic microorganisms to colonize new hosts and alter physiological processes has presented challenges. Therefore, the inventors sought to develop a method for more reliably colonizing human surfaces (e.g., the gastrointestinal tract or skin). The method of the present invention is based on the discovery of a technique for reliably performing microbiome transplantation. Briefly, commensal strains isolated from a human subject are cultured, transformed with heterologous polynucleotides to express heterologous proteins to produce therapeutic strains, and then administered in an engineered form to the same or a different human subject (e.g., autologous or allogeneic microbiome transplantation). Herein, the inventors demonstrate that long-term colonization and effective functional alterations in the gastrointestinal microbiome can be achieved by using native bacteria from the host as vectors to introduce new genes and functions into the luminal environment. Previous resistance to using this method has been attributed to the hypothesized difficulty of culturing and modifying native bacteria. By using native bacteria instead of laboratory strains, the inventors employ host cells that are already adapted to the host's luminal environment. This allows the engineered native bacteria (ENB) to establish and induce functional changes in the CR-WT host.

[0031] We identified, cultured, and isolated a tractable, naturally occurring bacterium from a CR-WT host, genetically modified it with genes theorized to confer beneficial functions, and then reintroduced ENB into the CR-WT host. Thus, ENB is already adapted to the luminal environment. In our preliminary studies, we used naturally occurring Escherichia coli isolated from mouse feces. Bacterial engineering of E. coli can be performed by almost any laboratory with very few resources. Although E. coli is a common naturally occurring bacterium, many researchers have assumed that it is not a good colonizer due to considerable disappointment with experimental strains. However, using our approach described herein, we successfully created engineered bacterial host cells that can be transformed to express heterologous polynucleotides and deliver therapeutic polypeptides to mammals. This strategy addresses the problem of high variability in colonization of many different hosts by a single specific strain. We demonstrate that engineered commensal bacteria isolated from the feces of a single mouse can be successfully stably colonized in the mouse gastrointestinal tract.

[0032] In our studies in CR-WT mice, we modified luminal bile and serum bile acids using our engineered native bacteria (ENB) to knock in bile salt hydrolase (BSH), a bacterial enzyme thought to deconjugate luminal bile acids (BAs) and affect multiple host physiological processes, including metabolism. We were surprised to discover that, in addition to affecting metabolism, activation of BSH in the intestinal lumen further influenced behavior and cognition. Previous studies have demonstrated a link between BAs, neuroinflammation, and cognition, and the results described herein are consistent with the conclusion that BAs are mediators of the microbiome-gut-brain axis.

[0033] By knocking in genes (e.g., bile salt hydrolase (BHS)) into the gut microbiome, we can reduce neuroinflammation. By alleviating neuroinflammation, we can treat a host of pathophysiological problems, including, but not limited to, obesity, type 2 diabetes, traumatic brain injury, dementia, stroke, and certain encephalopathies. We show herein that we can improve cognition in mice with diet-induced obesity-induced neuroinflammation.

[0034] 1. Methods for preparing engineered native / symbiotic bacteria Methods for preparing therapeutically suitable engineered native or commensal bacteria for transformation with heterologous polynucleotides are straightforward.

[0035] A microbiome sample is obtained from a patient. The microbiome sample may be derived from any bacterial population stably colonized on a surface or in a cavity of an individual. Microbial communities with stably colonized bacterial communities can be found on any site of the body exposed to the environment, such as the skin, nasopharynx, oral cavity, respiratory tract, gastrointestinal tract, and / or female reproductive tract. Thus, in some embodiments, a microbiome sample is obtained by swabbing, flushing, or biopsy of the skin, nasopharyngeal cavity, or oral cavity (e.g., cheek, tongue, gums, or throat), respiratory tract, gastrointestinal tract, and / or genitourinary tract. In some embodiments, a microbiome sample is obtained from a fecal sample. In some embodiments, a microbiome sample is obtained from a tissue biopsy (e.g., obtained during endoscopy, scraping biopsy, or punch biopsy). In some embodiments, a microbiome sample is obtained from a tissue surface (e.g., by swabbing or flushing with a solution). If desired, samples can be collected using a range of methods, including, but not limited to, cultures of bodily fluids (e.g., saliva, mucus, urine, stool, or breath), surface biopsies (e.g., mucosal biopsies of the gastrointestinal tract, oral / pharyngeal / nares biopsies, or genitourinary tract biopsies, skin biopsies), swabs (e.g., skin, anal / rectal, buccal / mouth, or eye), and / or pathological specimens (e.g., cancerous tissue, amputated limbs, or inflamed organs). A microbiome sample contains a sufficient number of cells to initiate one or more in vitro cultures for isolation, e.g., at least or at least about 1, 10, 100, 1000, 1 x 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , or 1 × 10 15 Contains bacterial cells.

[0036] A microbiome sample is homogenized, and bacterial cells from the homogenate are cultured on solid agar media to isolate the bacterial cells and then culture a substantially homogenous population of native or commensal bacterial cells for transformation with a heterologous polynucleotide. Using techniques known in the art, the homogenized sample is streaked onto a selective or specified solid microbial medium depending on the species of commensal or native bacteria to be isolated and cultured. Common bacterial genera found in the human microbiome that can be isolated and transformed to express a heterologous polynucleotide include, for example, Bacteroides, Clostridium, Streptococcus, Lactococcus, Eubacterium lectare, Escherichia coli, Enterobacter species, Klebsiella species, Bifidobacterium, Staphylococcus, Lactobacillus, Veillonella, Haemophilus, Moraxella, Corynebacterium, and Propionibacterium. Bacteroides species, previously considered the most common and abundant bacterial genus in the gastrointestinal tract, have been reclassified into five genera: Alistipes, Prevotella, Paraprevotella, Parabacteroides, and Odoribacter (Rajilic-Stojanovic et al., FEMS Microbiol Rev. 2014;38:996-1047). Depending on the needs, MacConkey lactose agar or violet red bile dextrose agar can be used to isolate and culture Escherichia coli cells. De Man-Rogosa-Sharpe agar can be used to isolate and culture Lactobacillus species cells. Bile esculin agar can be used to isolate and culture Enterococcus species cells. Wilkins-Challgren anaerobe agar can be used to isolate and culture Bacteroides species cells. TPY medium can be used to isolate and culture Bifidobacterium species. For Lactococcus species, BM9 or GM17c medium can be used.Bacterial species that commonly colonize the human microbiome are described, for example, by the Human Microbiome Project Consortium in Nature. (2012) June 13;486(7402):207-14 and Lloyd-Price, Genome Med. 2016 Apr 27;8(1):51.

[0037] Bacterial species commonly found in the human colon and which can be substantially isolated for transformation with a heterologous polynucleotide include, for example, Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Enterococcus faecalis, Escherichia coli, Enterobacter spp., Klebsiella spp., Bifidobacterium bifidum, Staphylococcus aureus, Lactobacillus spp., Clostridium perfringens, Proteus mirabilis, Clostridium tetani, Clostridium septicum, Pseudomonas aeruginosa, and the like. aeruginosa, Salmonella enterica, Faecalibacterium prausnitzii, Peptostreptococcus spp. and / or Peptococcus spp.

[0038] Bacterial species commonly found in human stool and that can be substantially isolated for transformation with a heterologous polynucleotide include, for example, Escherichia coli, Prevotella copri, Alistipes putredinis, and / or Bacteroides vulgatus.

[0039] Skin sites are colonized primarily by bacterial genera Corynebacterium, Propionibacterium, and / or Staphylococcus, which can be isolated and transformed to express a heterologous polynucleotide. Bacterial species commonly found on human skin that can be substantially isolated for transformation with a heterologous polynucleotide include, for example, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium species, Acinetobacter johnsonii, and / or Pseudomonas aeruginosa.

[0040] Bacterial species commonly found in the human oral cavity and that can be substantially isolated for transformation with a heterologous polynucleotide include, for example, Streptococcus (e.g., Streptococcus mitis), Haemophilus, Prevotella, Rothia mucilaginosa, and / or Corynebacterium matruchotii.

[0041] The major resident bacteria of the stomach that can be substantially isolated for transformation with a heterologous polynucleotide include Streptococcus, Staphylococcus, Lactobacillus, Helicobacter and / or Peptostreptococcus.

[0042] Bacterial species commonly found in the human vagina and that can be substantially isolated for transformation with a heterologous polynucleotide include, for example, Lactobacillus (e.g., L. crispatus, L. iners, L. jensenii, or L. gasseri), Gardnerella, and / or Prevotella.

[0043] The enrichment of the infant gut microbiome with commensal organisms such as Bacteroides, Parabacteroides, Clostridium, Lactobacillus, Bifidobacterium, and / or Faecalis prausnitzii contributes to numerous determinants of a healthy microbiome. These bacterial species can be transformed to express heterologous polynucleotides.

[0044] The candidate colony is restreaked on at least a second solid agar culture medium to substantially isolate it from contaminating strains. These purified isolates can be stored as cryogenic stocks. The purified strains are then subjected to testing to confirm their genus / species identity, the absence of pathogenic toxins, and susceptibility to clinically used antibiotics. For example, PCR and Sanger sequencing of all or part of the ribosomal 16S DNA sequence can be performed to confirm the genus / species identity.

[0045] The method eliminates or selects for commensal or natural bacterial colonies that express a pathogenic toxin. In some embodiments, the bacterial cells are confirmed to not express any known or selected pathogenic toxins. In some embodiments, the bacterial cells are identified to not express any known or selected pathogenic toxins, such as AB toxin, alpha toxin, anthrax toxin, botulinum toxin, cereulide, cholesterol-dependent cytolysin, Clostridial cytotoxin family, Clostridium botulinum C3 toxin, Clostridium difficile toxin A, Clostridium difficile toxin B, Clostridial enterotoxin, Clostridium perfringens alpha toxin, Clostridium perfringens beta toxin, Cry1Ac, Cry6Aa, Cry34Ab1, delta endotoxin, diphtheria toxin, enterotoxin, enterotoxin type B, erythrogenic toxin, exfoliatin, fragilisin, hemolysin E, heat-labile enterotoxin, thermotoxin, erythrotoxic to ... It is confirmed that the virus does not express one or more pathogenic toxins selected from the group consisting of: qualitative enterotoxins, hemolysins, HrpZ family, leukocidin, listeriolysin O, Panton-Valentine leukocidin, intact pathogenicity island, phenol-soluble modulin, pneumolysin, pore-forming toxins, Pseudomonas exotoxins, pyocyanin, anti-eukaryotic Rhs toxins, RTX toxins, Shiga toxins, Shiga-like toxins, Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin, Staphylococcus aureus delta toxin, streptolysin, tetanolysin, tetanospasmin, toxic shock syndrome toxin, tracheal cytotoxin, and / or Vero cytotoxin.

[0046] The method further selects for commensal or natural bacterial colonies that demonstrate sensitivity or susceptibility (e.g., lack of resistance) to clinically used antibiotics. In some embodiments, the bacterial cells are resistant to a macrolide antibiotic (e.g., azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylocine, or roxithromycin), a rifamycin (e.g., rifampicin (or rifampin), rifamycin), or a rifamycin (e.g., rifamycin). butin, rifapentine, rifalazil, or rifaximin), polymyxins (e.g., polymyxin B or polymyxin E (colistin)), quinolone antibiotics (e.g., nalidixic acid, ofloxacin, levofloxacin, ciprofloxacin, norfloxacin, enoxacin, lomefloxacin, grepafloxacin, trovafloxacin, sparfloxacin, temafloxacin, moxifloxacin, gatifloxacin, or gemifloxacin), beta-lactams (e.g., penicillin, cloxacin, cyclosacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, or piperacillin), aminoglycosides (e.g., amikacin, gentamicin, neomycin, streptomycin, or tobramycin), cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cefaclor, cefoxitin, cefprozil, cefuroxime, loracillin, beffu, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, cefepime, or ceftobiprole), monobactams (e.g., aztreonam, tigemonam, nocardicin A, or tabtoxin β-lactam), carbapenems (e.g., biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, or thienamycin), and / or tetracyclines (e.g., tetracycline, chlortetracycline,The bacterial cells are identified as having sensitivity or susceptibility (e.g., lack of resistance) to one or more antibiotic agents selected from the group consisting of oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, or tigecycline. Generally, transformation of a purified natural bacterial colony with a heterologous polynucleotide confers antibiotic resistance to one or more antibiotic agents used in selection of the transformed bacterial cells, e.g., resistance to kanamycin, chloramphenicol, carbenicillin, hygromycin, and / or trimethoprim.

[0047] Isolated colonies, confirmed to not express known pathological toxins and not susceptible to clinically important antibiotic agents, are transformed with one or more polynucleotides encoding one or more proteins heterologous to the bacterium and / or intended host. In some embodiments, a heterologous protein, such as a fluorescent protein, is used for detection. In some embodiments, the heterologous protein is a therapeutic polypeptide, as described in more detail below.

[0048] Isolated, substantially homogeneous colonies of native / commensal bacteria can be transformed using techniques known in the art. Such techniques are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Edition (2012). Clinical microbiology taxonomic manuals for guiding the selection and identification of bacterial species of interest include, for example, Medical Microbiology, 8th Edition, Murray, Rosenthal, and Pfaller, Elsevier, 2015; and Medical Microbiology: A Guide to Microbial Infections: Pathogenesis, Immunity, Laboratory Investigation and Control, 19th Edition, Barer, Irving, Swann, and Perera, Elsevier, 2018.

[0049] The isolated population of native / symbiotic bacteria is transformed, e.g., genetically modified, to express one or more heterologous polypeptides of interest, e.g., one or more therapeutic polypeptides listed in Table 1 and / or a detectable protein, such as a fluorescent protein.

[0050] The polynucleotide encoding the heterologous polypeptide may be introduced into a vector, preferably an expression vector. A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. An expression vector contains one or more regulatory sequences that direct the expression of a gene to which it is operably linked. "Operably linked" intends that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include controllable transcription promoters, operators, enhancers, transcription terminators, and other expression control elements, such as translation control sequences (e.g., Shine-Dalgarno consensus sequences, start and stop codons). These regulatory sequences are expected to vary, for example, depending on the host cell used.

[0051] Polynucleotides encoding heterologous polypeptides may be codon biased for improved expression in the native / symbiotic bacterial host cells. Preferred codon usage for the genus and species of the isolated and transformed symbiotic or native bacterial host cells described herein is known and is listed in available codon usage databases, for example, at kazusa.or.jp / codon / .

[0052] Vectors can replicate autonomously in host cells (episomal vectors) or can integrate into the genome of a host cell and replicate along with the host genome (non-episomal mammalian vectors). Integrating vectors typically contain at least one sequence homologous to a bacterial chromosome, allowing recombination between homologous DNA in the vector and the bacterial chromosome. Integrating vectors may also contain bacteriophage or transposon sequences. Episomal vectors or plasmids are circular double-stranded DNA loops into which additional DNA segments can be ligated. When using recombinant DNA techniques, plasmids capable of stable maintenance in a host are generally the preferred form of expression vector. Useful exemplary bacteriophage recombination systems are described, for example, in Nafissi et al., Appl Microbiol Biotechnol. 2014 Apr;98(7):2841-51. Additional useful bacteriophage delivery systems are described, for example, in U.S. Patent Publication No. 2016 / 0367701. Symbiotic growth can have a detrimental effect on plasmid maintenance. If desired, facilitating or promoting the maintenance or retention of plasmids in the transformed symbiotic / native bacterial host cell can be achieved using methods known in the art.Such plasmid retention or maintenance strategies include, but are not limited to, partitioning systems (e.g., parABS; see, e.g., Yamaichi et al., Proc Natl Acad Sci U S A. (2000) 97(26):14656-61; Youngren et al., J Bacteriol. 2000 July;182(14):3924-8; Dubarry et al., J Bacteriol. 2006 February;188(4):1489-96; and Hanai et al., J. Biol. Chem. (1996) 271:17469-17475), or toxin-antitoxin modules or systems (e.g., ccdAB, hok-sok; see, e.g., Fernandez-Garcia et al., Toxins (Basel). (2016) July 20;8(7). pii: E227); Fang et al., Appl. Environ. Microbiol. (2008) 74(10):3216-3228; Lobato-Marquez et al., Front Mol Biosci. 2016 Oct. 17;3:66; Zielenkiewicz et al., J. Bacteriol. (2005) 187(17):6094-6105; Leplae et al., Nucleic Acids Research, (2011) 39(13) 5513-5525 and Grady et al., Molecular Microbiology (2003) 47(5):1419-1432).

[0053] Regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of a nucleotide sequence only under specific environmental conditions. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating downstream (3') transcription of a coding sequence (e.g., a structural gene) into mRNA. A promoter is expected to have a transcription initiation region, which is usually located proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters may also have a second domain called an operator, which may overlap an adjacent RNA polymerase binding site where RNA synthesis begins. Operators allow negatively regulated (inducible) transcription because gene repressor proteins can bind to the operator and thereby inhibit transcription of a specific gene. Constitutive expression can also occur in the absence of negative regulatory elements such as operators. In addition, positive regulation can be achieved by gene activator protein binding sequences, which, if present, are usually proximal (5') to the RNA polymerase binding sequence. Exemplary useful regulator / promoter systems for expressing heterologous polynucleotides in transformed native / symbiotic bacterial cells include, but are not limited to, XylS / Pm (wild-type), XylS / PmML1-17 (Pm variant), LacI / PT7lac, LacI / Ptrc, and / or AraC / PBAD. See Balzar et al., Microbial Cell Factories 2013, 12:26.

[0054] An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in E. coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Therefore, expression control can be either positive or negative, so that transcription can be either enhanced or reduced. Other examples of positive and negative regulatory elements are well known in the art. Various promoters that can be included in the protein expression system include, but are not limited to, the T7 / LacO hybrid promoter, the trp promoter, the T7 promoter, the lac promoter, the p6 promoter, and the bacteriophage lambda promoter. Any suitable promoter, including the native promoter or a heterologous promoter, can be used to carry out the present invention. The heterologous promoter may be constitutively active or inducible. Non-limiting examples of heterologous promoters are provided in U.S. Patent No. 6,242,194 to Kullen and Klaenhammer.

[0055] A "constitutive promoter" refers to a promoter capable of facilitating the continuous transcription of a coding sequence or gene under its control and / or operably linked thereto. Exemplary useful constitutive promoters include, but are not limited to, BBa_J23100, constitutive E. coli σ S promoters (e.g., osmY promoter (standard biological part names in the International Genetically Engineered Machine (iGEM) registry are BBa_J45992; BBa_J45993)), constitutive E. coli σ 32 Promoter (e.g., htpG heat shock promoter (BBa_J45504)), constitutive E. coli σ 70Promoters (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate-sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa _M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), constitutive Bacillus subtilis σ A Promoters (e.g., promoter veg(BBa_K143013), promoter 43(BBa_K143013), PliaG(BBa_K823000), PlepA(BBa_K823002), Pveg(BBa_K823003)), constitutive Bacillus subtilis σ B promoters (e.g., promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)), Salmonella promoters (e.g., Pspv2 from Salmonella (BBa_K112706), Pspv from Salmonella (BBa_K112707)), bacteriophage T7 promoters (e.g., T7 promoters (BBa_I712074; BBa_I719005; BBa_J 34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253), and / or bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)).

[0056] Examples of useful inducible promoters include, but are not limited to, the FNR promoter, the ParaC promoter, the ParaBAD promoter, the propionic acid promoter, and / or the PTetR promoter.

[0057] To maintain the ability for long-term or persistent colonization of mammals (e.g., the ability to be successfully reintroduced into a mammalian microbiome), populations of native / commensal bacteria transformed to express one or more heterologous polypeptides are not adapted to a laboratory or in vitro culture environment. ENB are cultured in vitro in an experimental environment to minimize disruption as much as possible. In some embodiments, ENB are cultured in vitro in an experimental environment outside of the donor subject for 30 days or less, e.g., 25, 20, 15, 10 days or less, prior to administration to a recipient subject. In some embodiments, the total in vitro growth time of ENB is about 14 days or less, e.g., 13, 12, 11, 10, 9, 8, 7 days or less, between collection from the donor subject and administration to the recipient subject. Calculations of such in vitro growth or culture time generally do not include the time the bacterial cells are stored (e.g., cryopreserved or lyophilized), but include the time it takes to transform or introduce one or more heterologous polynucleotides.

[0058] In some embodiments, the native / symbiotic host cell (i) is capable of metabolizing one or more carbohydrates selected from the group consisting of sucrose, xylose, d-maltose, N-acetyl-d-glucosamine, d-galactose, and d-ribose; (ii) utilizes both glycolytic and gluconeogenic substrates; (iii) is non-motile (e.g., has a non-functional flagellum, e.g., due to a mutation in the flhDC operon); (iv) is capable of producing ribose-5-phosphate; (v) is capable of growing in defined media lacking vitamin B12 (cyanocobalamin) (e.g., vitamin B12 prototrophy has been demonstrated); (vi) expresses UDP-glucose-4-epimerase and / or glycosyltransferase; (vii) is capable of consuming tryptophan. (viii) contain multiple copies of the gene encoding the β subunit of the propionate CoA-transferase gene; (ix) express capsular polysaccharide (CPS) 4 (CPS4); (x) express an rnf-like oxidoreductase complex; (xi) catabolize tryptophan to indole and other indole metabolites, e.g., indole-3-propionate and indole-3-aldehyde; and / or do not produce any substances that induce double-stranded DNA breaks, e.g., do not have genomic islands encoding large modular non-ribosomal peptide and polyketide synthases, do not express hybrid peptide-polyketide genotoxic substances, and / or do not have an active clbA gene. Genotypes and phenotypes that contribute to the ability of commensal bacteria to stably colonize on or in mammals are described, for example, in Lozupone et al., Genome Res (2012) 22:1974-1984; Leatham et al., Infect. Immun. (2005) 73(12):8039-8049; Miranda et al., Infect. Immun. (2004) 72(3):1666-1676; Leatham et al., Infect. Immun. (2009) 77(7):2876-2886 and Goodman et al., Cell Host Microbe. 2009 Sep 17;6(3):279-289.Others have found that laboratory-adapted E. coli strains, such as Nissle 1917, induce DNA double-strand breaks. See, e.g., Nougayrede et al., Science (2006) 313(5788):848-851; and Olier et al., Gut Microbes. (2012) 3(6):501-509. Populations of native / symbiotic bacteria transformed to express one or more heterologous polypeptides can be cryopreserved or lyophilized for long-term storage.

[0059] 2. Conditions to be treated, mitigated and / or prevented Depending on the heterologous polynucleotide expressed by the ENB and the route of administration, the ENBs described herein find use in treating or preventing a number of disease states, as summarized in Table 1.

[0060] For example, in some embodiments, a population of native / probiotic bacterial cells transformed to express a bile salt hydrolase (e.g., from the genus Lactobacillus or Bifidobacterium) can be administered to the gastrointestinal tract, e.g., orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with obesity / type 2 diabetes, chronic kidney disease, cognitive decline / deficiency (e.g., due to traumatic brain injury, dementia, stroke, hepatic encephalopathy, infantile anoxic brain injury), hypercholesterolemia, male infertility, female infertility, and C. difficile infection. In some embodiments, the heterologous polynucleotide encodes a bile salt hydrolase having at least or at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to GenBank: ACL98194.1 (BSH from Lactobacillus salivarius); NCBI Reference Sequence: YP_193782.1 (bshA from Lactobacillus acidophilus); NCBI Reference Sequence: YP_193954.1 (bshB from Lactobacillus acidophilus); or Gene ID: 31838777 (RBL67 conjugated bile salt hydrolase D805_RS01800 from Bifidobacterium thermophilum).

[0061] In some embodiments, a population of natural / commensal bacterial cells transformed to express mammalian (e.g., human) sulfotransferase family 2A member 1 (SULT2A1) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with obesity / type 2 diabetes, hypercholesterolemia, non-alcoholic steatohepatitis (NAFLD), and dementia / cognitive decline.

[0062] In some embodiments, a population of native / commensal bacterial cells transformed to express mammalian (e.g., human) NAPE-hydrolyzing phospholipase D (NAPEPLD), FGF1, FGF15, FGF19 and / or glucagon (GLP-1) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with obesity / type 2 diabetes.

[0063] In some embodiments, one or more populations of natural / commensal bacterial cells transformed to express mammalian (e.g., human) IL-10, TGFβ, IL-27 dimer and / or anti-TNFα antibody can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with an autoimmune disease, such as ulcerative colitis, Crohn's disease, type 1, or autoimmune diabetes.

[0064] In some embodiments, a population of native / commensal bacterial cells transformed to express mammalian (e.g., human) trefoil factors (e.g., TFF1, TFF2, and / or TFF3) or peptidase inhibitor 3 (PI3) or elafin (Serpina1c) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with inflammatory diseases, such as oral mucositis, ulcerative colitis, and Crohn's disease.

[0065] In some embodiments, a population of native / commensal bacterial cells transformed to express Vibrio vulnificus flagellin B can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with cancer, for example, cancer of the gastrointestinal tract, for example, oral cancer, esophageal cancer, stomach cancer, colon cancer, or rectal cancer.

[0066] In some embodiments, a population of native / commensal bacterial cells transformed to express Nostoc ellipsosporum cyanovirin-N can be administered to the gastrointestinal tract, for example orally and / or rectally, and / or to the genitourinary tract to alleviate, alleviate, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with HIV.

[0067] In some embodiments, a population of native / commensal bacterial cells transformed to express Actinobacillus actinomycetemcomitans dispersin B (DspB) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with Pseudomonas aeruginosa infection.

[0068] In some embodiments, a population of natural / commensal bacterial cells transformed to express microcin J25 (MccJ25) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with Salmonella enterica infection.

[0069] In some embodiments, a population of native / commensal bacterial cells transformed to express cholylglycine hydrolase and / or cholic acid 7alpha-dehydroxylase can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with Clostridium difficile infection.

[0070] In some embodiments, a population of native / commensal bacterial cells transformed to express Akkermansia muciniphila Amuc_1100* can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with non-alcoholic steatohepatitis (NAFLD) and / or aging / senescence.

[0071] In some embodiments, a population of native / commensal bacterial cells transformed to express a bile acid-CoA:amino acid N-acyltransferase (BAAT) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with malnutrition.

[0072] In some embodiments, a population of native / probiotic bacterial cells transformed to express mammalian (e.g., human) lactase can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with lactose intolerance.

[0073] In some embodiments, a population of native / commensal bacterial cells transformed to express mammalian (e.g., human) phenylalanine hydroxylase can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with phenylketonuria.

[0074] In some embodiments, a population of native / probiotic bacterial cells transformed to express a mammalian (e.g., human) alcohol dehydrogenase can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with alcohol intolerance / toxicity.

[0075] In some embodiments, a population of native / probiotic bacterial cells transformed to express Aspergillus niger derived prolyl endoprotease (AN-PEP) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, relieve, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with celiac disease.

[0076] In some embodiments, a population of native / commensal bacterial cells transformed to express 7-alpha-hydroxysteroid dehydrogenase (hdhA) and / or 7 beta-hydroxysteroid dehydrogenase (7β-HSDH; EC 1.1.1.201) can be administered to the gastrointestinal tract, for example orally and / or rectally, to alleviate, alleviate, reduce, inhibit, ameliorate, and / or prevent one or more symptoms caused by or associated with traumatic brain injury, dementia / cognitive decline, hepatic encephalopathy, and infant anoxic brain injury.

[0077] The mammal subjected to such therapy may be symptomatic or asymptomatic. The mammal may have a family history or a determined genetic risk for the disease state. The mammal may be an adult, juvenile, child, or infant.

[0078] Upon delivery to the gastrointestinal tract, engineered native bacteria (ENB) do not significantly alter the gastrointestinal microbiome, e.g., the microbiome of the terminal ileum, regardless of the presence or absence of a therapeutic polypeptide. This can be confirmed, for example, by analysis, e.g., sequencing of the ribosomal 16S DNA of the microbiome, before and after ENB administration. Thus, in some embodiments, a subject is selected or identified as being within a class of subjects in need of such therapy, and selection or identification can be made by clinical or diagnostic evaluation.

[0079] [Table 1A]

[0080] [Table 1B]

[0081] [Table 1C]

[0082] [Table 1D]

[0083] [Table 1E]

[0084] [Table 1F]

[0085] [Table 1G]

[0086] [Table 1H]

[0087] 3. Formulation and Administration Natural / probiotic bacteria transformed to express a heterologous polynucleotide (ENB) can be formulated into bacterial compositions for administration to humans and other mammalian subjects in need thereof. Generally, the bacterial composition is combined with additional active and / or inactive materials to produce a final product, which may be in the form of a single dosage unit or in a multi-dose format. In some embodiments, the bacterial composition is comprised of one or more ENB populations described herein. In some embodiments, the bacterial composition is comprised of one or more ENB populations and one or more prebiotics.

[0088] The composition may include different types of carriers depending on whether it is administered as a solid or liquid. ENB compositions can be administered orally, intravaginally, intrarectally, topically (e.g., to the eye or conjunctiva), intratumorally, via vesicle instillation (e.g., to the bladder), intralesionally, intranasally, topically, or bucally. In various embodiments, the composition can be delivered, for example, via food, drink, capsule, tube feeding, enema, suppository, infusion, continuous infusion, via localized perfusion directly bathing target cells, via catheter, via irrigation, in a lipid composition (e.g., liposomes), as an aerosol, or by any other method or combination of the foregoing that would be expected to be known to those skilled in the art (see, e.g., Lloyd V. Allen, Jr., Remington: The Science and Practice of Pharmacy, 22nd ed., 2012, Pharmaceutical Press, which is expressly incorporated herein by reference in its entirety).

[0089] In some embodiments, the composition comprises at least one prebiotic carbohydrate. "Carbohydrate" refers to a sugar or a polymer of sugar. The terms "sugar," "polysaccharide," "carbohydrate," and "oligosaccharide" can be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one hydroxyl group for each carbon atom of the molecule. Carbohydrates generally have the molecular formula C n H 2n O n Carbohydrates may be monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. Most basic carbohydrates are monosaccharides, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two monosaccharides joined together. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, oligosaccharides contain three to six monosaccharide units (e.g., raffinose or stachyose), while polysaccharides contain six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and / or cellulose. Carbohydrates may contain modified sugar units, e.g., 2'-deoxyribose in which the hydroxyl group has been removed, 2'-fluororibose in which the hydroxyl group has been replaced with fluorine, or N-acetylglucosamine, nitrogen-containing forms of glucose (e.g., 2'-fluororibose, deoxyribose, and / or hexose). Carbohydrates can exist in many different forms, e.g., conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and / or isomers.

[0090] In some embodiments, the composition comprises at least one lipid. "Lipid," as used herein, includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in all forms, such as free fatty acids. Fats, oils, and fatty acids may be saturated, unsaturated (cis or trans), or partially unsaturated (cis or trans). In some embodiments, the lipid is selected from the group consisting of lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (2 In another embodiment, the composition comprises at least one fatty acid selected from eicosenoic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and / or tetracosanoic acid (24:0). In another embodiment, the composition comprises at least one modified lipid, e.g., a lipid modified by cooking.

[0091] In some embodiments, the composition includes at least one supplemental inorganic substance or inorganic substance source. Examples of inorganic substances include, but are not limited to, chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and / or selenium. Suitable forms of any of the aforementioned inorganic substances include soluble inorganic salts, slightly soluble inorganic salts, insoluble inorganic salts, chelated inorganic substances, inorganic substance complexes, non-reactive inorganic substances such as carbonyl inorganic substances, and / or reduced inorganic substances, and combinations thereof.

[0092] In certain embodiments, the composition includes at least one supplemental vitamin and / or antioxidant. The at least one vitamin may be a fat-soluble vitamin or a water-soluble vitamin. Suitable vitamins include, but are not limited to, vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and / or biotin. Suitable forms of any of the foregoing include salts of vitamins, derivatives of vitamins, compounds having the same or similar activity as vitamins, and metabolites of vitamins.

[0093] In other embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include buffering agents, preservatives, stabilizers, binders, compression agents, lubricants, dispersion enhancers, disintegrants, flavoring agents, sweeteners, and / or coloring agents.

[0094] In another embodiment, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and / or calcium bicarbonate.

[0095] In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobial agents, such as parabens, chlorobutanol, and / or phenol.

[0096] If the formulation contains an anaerobic strain, the pharmaceutical formulation and excipients can be selected to prevent exposure of the strain to oxygen.

[0097] In other embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 ~C 18 Included are fatty acid alcohols, polyethylene glycols, polyols, sugars, or oligosaccharides, and combinations thereof.

[0098] In another embodiment, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, Sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and / or light mineral oil.

[0099] In other embodiments, the composition includes a dispersing aid as an excipient. Non-limiting examples of suitable dispersing agents include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and / or microcrystalline cellulose as a high HLB emulsifier surfactant.

[0100] In some embodiments, the composition comprises a disintegrant as an excipient. In other embodiments, the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches, such as corn starch, potato starch, pregelatinized and / or modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, or gums, such as agar, guar, locust bean, karaya, pectin, and / or tragacanth. In another embodiment, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include a combination of sodium bicarbonate and citric acid, and / or a combination of sodium bicarbonate and tartaric acid.

[0101] In another embodiment, the excipient comprises a flavoring agent. The flavoring agent can be selected from synthetic flavor oils and / or fragrances; natural oils; extracts from plants, leaves, flowers, and / or fruits; and combinations thereof. In some embodiments, the flavoring agent is selected from cinnamon oil; oil of wintergreen; peppermint oil; clove oil; hay oil; anise oil; eucalyptus; vanilla; citrus oils, such as lemon oil, orange oil, grape and / or grapefruit oil; and / or fruit essences such as apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and / or apricot.

[0102] In other embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and / or mixtures thereof (when not used as a carrier); saccharin and / or its various salts, such as the sodium salt; dipeptide sweeteners, such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia rebaudiana (stevioside); chloro derivatives of sucrose, such as sucralose; and / or sugar alcohols, such as sorbitol, mannitol, and sylitol. Hydrogenated starch hydrolysates and the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly its potassium salt (acesulfame-K) and / or its sodium and calcium salts, are also contemplated.

[0103] In some embodiments, the composition comprises a colorant. Non-limiting examples of suitable colorants include Food, Drug, and Cosmetic Colorants (FD&C), Drug and Cosmetic Colorants (D&C), and / or External Drug and Cosmetic Colorants (Ext. D&C). The colorant can be used as a dye or its corresponding lake.

[0104] In various embodiments, the mass fraction of an excipient or combination of excipients in a formulation is typically about 99% or less (but not zero), e.g., about 95% or less (but not zero), about 90% or less (but not zero), about 85% or less (but not zero), about 80% or less (but not zero), about 75% or less (but not zero), about 70% or less (but not zero), about 65% or less (but not zero), about 60% or less (but not zero), about 55% or less (but not zero), about 50% or less of the total mass of the composition. or 50% or less (but not zero), about 45% or less (but not zero), about 40% or less (but not zero), about 35% or less (but not zero), about 30% or less (but not zero), about 25% or less (but not zero), about 20% or less (but not zero), about 15% or less (but not zero), about 10% or less (but not zero), about 5% or less (but not zero), about 2% or less (but not zero), or about 1% or less (but not zero).

[0105] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and / or granules. Capsules typically include a core material containing the bacterial composition and a shell wall encapsulating the core material. In some embodiments, the core material includes at least one of a solid, a liquid, and / or an emulsion. In other embodiments, the shell wall material includes at least one of soft gelatin, hard gelatin, and / or a polymer. Suitable polymers include, but are not limited to, cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate, and sodium carboxymethylcellulose; acrylic acid polymers and / or copolymers such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammoniomethyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate (e.g., copolymers sold under the trade name "Eudragit"); vinyl polymers and / or copolymers such as polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and / or ethylene-vinyl acetate copolymer; and / or shellac (purified lac). In yet other embodiments, at least one polymer functions as a taste-masking agent.

[0106] Tablets, pills, etc. may be compressed, multi-compressed, multi-layered, and / or coated. The coating may be single or multiple. In one embodiment, the coating material comprises at least one sugar, polysaccharide, and / or glycoprotein extracted from at least one plant, fungus, and / or microorganism. Non-limiting examples include corn starch, wheat starch, potato starch, tapioca starch, cellulose, hemicellulose, dextran, maltodextrin, cyclodextrin, inulin, pectin, mannan, gum arabic, locust bean gum, mesquite gum, guar gum, karaya gum, ghatti gum, tragacanth gum, funori, carrageenan, agar, alginate, chitosan, or gellan gum. In some embodiments, the coating material comprises a protein. In another embodiment, the coating material comprises at least one of a fat and an oil. In another embodiment, at least one of the fats and oils melts at a high temperature. In yet another embodiment, at least one of the fats and oils is hydrogenated or partially hydrogenated. In one embodiment, at least one of the fats and oils is derived from a plant. In another embodiment, at least one of the fats and oils comprises at least one of a glyceride, a free fatty acid, and / or a fatty acid ester. In some embodiments, the coating material comprises at least one edible wax. The edible wax may be derived from an animal, an insect, or a plant. Non-limiting examples include beeswax, lanolin, bayberry wax, carnauba wax, and / or rice bran wax. Tablets and pills can further be prepared with an enteric coating.

[0107] Alternatively, powders or granules embodying the bacterial compositions disclosed herein can be incorporated into food products. In some embodiments, the food product is a drink for oral administration. Non-limiting examples of suitable drinks include fruit juices, fruit drinks, artificially flavored drinks, artificially sweetened drinks, carbonated drinks, sports drinks, liquid dairy products, shakes, alcoholic drinks, caffeinated drinks, or infant formula. Other suitable products for oral administration include aqueous and non-aqueous solutions, emulsions, suspensions, and / or solutions containing at least one suitable solvent, preservative, emulsifier, suspending agent, diluent, sweetener, colorant, and / or flavoring agent, and / or suspensions reconstituted from non-effervescent granules.

[0108] In some embodiments, the food product may be a solid food ingredient. Suitable examples of solid food ingredients include, but are not limited to, a food bar, a snack bar, a cookie, a brownie, a muffin, a cracker, an ice cream or ice cream bar, a yogurt or frozen yogurt bar.

[0109] In other embodiments, the compositions disclosed herein are incorporated into therapeutic foods. In some embodiments, therapeutic foods are ready-to-eat foods that optionally contain some or all essential macronutrients and micronutrients. In another embodiment, the compositions disclosed herein are incorporated into supplemental foods designed to be blended into existing meals. In one embodiment, the supplemental food or dietary supplement contains some or all essential macronutrients and micronutrients. In another embodiment, the bacterial compositions disclosed herein are blended with or added to existing foods to fortify the protein nutrition of the foods. Examples include staple foods (cereals, salt, sugar, cooking oil, or margarine), beverages (coffee, tea, soda, water, beer, liquor, or sports drinks), snacks, or confectionery and other foods.

[0110] In one embodiment, the formulation is filled into a gelatin capsule for oral administration. An example of a suitable capsule contains 10 (up to 100 mg) of lyophilized powder (e.g., 10 8 From 10 11 100 mg of bacterial cells), 160 mg of microcrystalline cellulose, 77.5 mg of gelatin, and 2.5 mg of magnesium stearate. 5 From 10 12 bacterial cells, e.g., 10 5 From 10 7 , 10 6 From 10 7 , or 10 8 From 10 10 Individual bacterial cells can be used, with concomitant adjustments made with excipients as needed. In alternative embodiments, enteric coated capsules or tablets or buffered or protective compositions can be used.

[0111] Bacterial compositions, with or without one or more prebiotics, are generally formulated for oral or gastric administration, typically to mammalian subjects. In certain embodiments, the compositions are formulated for oral administration in solid, semi-solid, gel, or liquid form, such as a pill, tablet, capsule, or lozenge. In some embodiments, such formulations contain or are coated with an enteric coating to protect the bacteria from the stomach to the small intestine, although spores generally resist the stomach and small intestine. In other embodiments, bacterial compositions, with or without one or more prebiotics, may be formulated with a germinant to enhance survival or efficacy. In still other embodiments, bacterial compositions may be formulated or administered with a prebiotic substance to enhance survival or efficacy. In some embodiments, bacterial compositions may be formulated or administered with a prebiotic substance to enhance survival or efficacy.

[0112] Bacterial compositions, with or without one or more prebiotics, may be formulated to be effective in a given mammalian subject in a single dose or over multiple doses. For example, a single dose may be substantially effective to reduce or increase a monitored symptom or biomarker of a targeted disease state, such as increasing insulin, increasing metabolism, increasing cognitive performance, or reducing inflammatory and / or autoimmune responses, in the mammalian subject to which the composition is administered. By substantially effective, it is meant that the presence of a monitored symptom or biomarker in the subject is reduced or increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99% after administration of the composition.

[0113] In some embodiments, the composition provides a single oral dose of at least or at least about 1 x 10 4 It is formulated to contain colony forming units of bacterial and / or fungal entities, and a single oral dose is typically about 1 x 10 4 or 1 x 10 4 , about 1×10 5 or 1 x 10 5 , about 1×10 6 or 1 x 10 6 , about 1×10 7 or 1 x 10 7 , about 1×10 8 or 1 x 10 8 , about 1×10 9 or 1 x 10 9 , about 1×10 10 or 1 x 10 10 , about 1×10 11 or 1 x 10 11 , about 1×10 12 or 1 x 10 12 , about 1×10 13 or 1 x 10 13 , about 1×10 14 or 1 x 10 14 , about 1×10 15 or 1 x 10 15 CFU of bacterial entities, or 1 x 10 15It is expected that the composition will contain more than 1×10 CFU of bacterial entities. For example, the concentration of cells of a given strain or collection of all strains, if known, may be greater than, for example, 1×10 per gram of composition or per administered dose. 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 pieces, or 1 x 10 15 More than one viable bacterial entity (e.g., CFU).

[0114] In some formulations, the composition contains at least or at least about 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% bacterial cells by weight. In some formulations, the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 grams by weight.

[0115] 4. Kit Further provided are kits comprising one or more containers containing one or more isolated populations of engineered naturally occurring bacteria (ENB) described herein or one or more compositions comprising one or more isolated populations of engineered naturally occurring bacteria. In various embodiments, the container may contain multiple, integrated portions or doses of a composition comprising a population of ENB transformed to express the same one or more heterologous polynucleotides. In various embodiments, the container may contain multiple, integrated portions or doses of a composition comprising a population of ENB transformed to express different one or more heterologous polynucleotides. In various embodiments, the container contains an edible composition, e.g., a food, beverage, or capsule. In various embodiments, the container contains a integrated volume of a buffered solution or suspension comprising a population of ENB, e.g., a population of ENB transformed to express the same one or more heterologous polynucleotides. In various embodiments, the container contains, e.g., a integrated dose of lyophilized ENB and a buffered solution for reconstitution. [Example]

[0116] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0117] Example 1 Exemplary Methods for Preparing Engineered Probiotic Bacteria Sample collection. For human patients, mucosal biopsies of the duodenum, ileum, and colon were collected during endoscopy and suspended in 1.8 mL of 1x PBS in a 15 mL conical tube at room temperature. Within 8 hours, the samples were transferred to sterile 2 mL screw-top microcentrifuge tubes and sterile 1.5 mm diameter chromium-coated steel beads were added.

[0118] For mice, feces were collected by placing them in a sterile 1 L polypropylene cup until 2-5 whole fecal pellets were excreted. The mice were returned to their home cages, and fecal pellets were collected using sterile forceps and placed into pre-weighed, sterile 2 mL screw-top microcentrifuge tubes. Tissue samples were collected after euthanasia by carbon dioxide and cervical dislocation and placed into pre-weighed, sterile 2 mL screw-top microcentrifuge tubes. The following workup was the same for both tissue and feces. Within 2 hours, the centrifuge tubes were weighed again, and 1 mL of sterile deionized water was added to each tube, followed by the addition of sterile 1.5 mm diameter chrome beads.

[0119] Sample Processing. The samples were homogenized using a BioSpec Bead Beater Mini 24 at 3800 RPM for 1 minute, cooled on ice for approximately 1 minute, homogenized at 3800 RPM for an additional minute, and cooled on ice again for approximately 1 minute. The chrome-steel beads were removed by running a rare earth magnet down the side of the tube to remove the beads.

[0120] For human tissue samples, the resulting homogenate was placed in an Eppendorf 5430R centrifuge at 14,000 RPM for 1 minute to pellet the cells and tissue. 1.5 mL of the resulting supernatant was transferred to a new, clean, screw-top microcentrifuge tube and stored at room temperature. The pellet was resuspended in the remaining supernatant by vortexing the microcentrifuge tube for 30-60 seconds.

[0121] For mouse samples, the homogenized samples were then used directly.

[0122] Strain isolation. For each homogenized sample, 200 μL was dispensed onto a disposable 10 cm Petri dish containing 25 mL of hardened selective agar medium and spread using 10-50 sterile 1 mm diameter glass beads until the water evaporated or was absorbed into the medium. The sample was then diluted 10-fold, and 100 μL of the diluted sample was then plated onto the same plate. This dilution plating was performed up to 1000-fold dilutions.

[0123] For the isolation of E. coli strains, the selective agar medium was either MacConkey lactose agar ("MacConkey") or Eosin-Methylene Blue lactose agar ("EMB"). For the isolation of Lactobacillus strains, the selective agar medium was De Man-Rogosa-Sharpe agar ("MRS").

[0124] For the isolation of E. coli, the resulting agar plates were incubated at 37°C in a humidified room air (i.e., aerobic) environment. For the isolation of Lactobacillus species, the resulting agar plates were incubated at 37°C in sealed jars containing AnaeroPack (Mitsubishi Gas Chemical Company of America, Inc.). After approximately 12–24 h, when colonies (and their medium-specific phenotype, such as halos on MacConkey agar) became visible, candidate colonies were selected and manually streaked for isolation on the same agar medium as the source plate. After incubating these agar plates under the same conditions as the original plates, colonies isolated by at least a 3 mm zone of no growth were selected and grown in sterile 16 mm x 160 mm glass culture tubes containing 1 mL of liquid growth medium. For E. coli, the liquid medium was Lysogen liquid medium containing 0.2% w / v glucose and 5 g / L NaCl. For Lactobacillus strains, the liquid medium was De Man-Rogosa-Sharpe medium, and the tubes were sealed with rubber stoppers. Cultures were grown by shaking in an orbital shaker at 37°C until they became turbid, at which point 0.5 mL of culture was transferred to screw-top cryovials containing glycerol (0.05 mL for Lactobacillus and 0.1 mL for E. coli), mixed by trituration, and stored at 80°C.

[0125] Strain Identification. Cells from the remaining liquid culture were transferred to a sterile 1.7 mL microcentrifuge tube and collected by centrifugation at 14,000 RPM for 1 minute. The supernatant was aspirated and discarded, and the cell pellet was resuspended in 50 μL of sterile deionized water.

[0126] Ribosomal 16S DNA was amplified by PCR using primers specific for the V3-V4 region with Phusion DNA polymerase (New England Biolabs). Cells suspended in water were used directly as template material for a 10-fold dilution starting from the concentrated cell slurry. The total reaction volume was 25 μL. After cycling, 5 μL of each reaction was analyzed by elution on a 1% w / v 0.5x TAE agarose gel with SYBR Safe DNA stain (Invitrogen). Reactions showing an approximately 450 bp product and no substantial primer dimer or nonspecific product bands were identified, and the remaining 20 μL of PCR solution was purified using a silica spin column kit (Invitrogen or New England Biolabs). The resulting DNA was quantified by UV-visible spectrophotometry and sequenced by Sanger dideoxy sequencing by a service provider (Eton).

[0127] Upon receiving sequencing results, chromatograms were manually inspected for strong, well-resolved fluorescent peaks. DNA base calls from these chromatograms were compiled and aligned against the NCBI non-redundant ("nr") database using BLAST. Strains were tentatively identified by the predominance of genus / species genomes with ≥97% identity to the sample sequence.

[0128] To confirm strain identity and uniquely identify strains from one another, regions of highly variable genes specific to the identities assigned by 16S sequencing were amplified and sequenced. In the case of E. coli, gyrB and fumC sequence alleles were used. Table 2 summarizes the strain identities of the different E. coli strains we isolated.

[0129] [Table 2]

[0130] Engineering the progenitor strain. E. coli MG1655 (a laboratory-adapted strain) was transformed with pSIM18, which encodes the lambda-Red recombination genes bet, exo, and gam under the control of a 42°C-inducible promoter, confers resistance to hygromycin, and can be eliminated from containing strains by nonselective growth at 37°C or higher. This strain, MG1655(pSIM18), was used to generate a number of additional derivative strains.

[0131] To confer constitutive expression of the marker gene green fluorescent protein (GFP), we fused the gfpmut2 gene to the promoter region for the E. coli gene rplN, as described by Zaslaver et al., Nat Methods. 2006 Aug;3(8):623-8, and used a sequence containing the aph kanamycin resistance-conferring gene for plasmid collection. This sequence was amplified by PCR with a 30-40 nucleotide overhang homologous to the bacteriophage lambda attachment site, attB. This PCR fragment was used for recombination with MG1655(pSIM18) to produce strain MG1655attB::[aphPrplN-gfpmut2]KmR. This strain fluoresces green under blue light, and its sequence was confirmed by Sanger dideoxy sequencing (SEQ ID NO: 1). Cultures of these cells were lysed with bacteriophage P1vir to produce the transducing phage P1vir(attB::[aph PrplN-gfpmut2]KmR).

[0132] To confer expression of heterologous genes, we constructed an expression cassette containing a hybrid Ptrc promoter sequence, an [aph PrhaB-ccdB] counterselection cassette, and a chloramphenicol acetyltransferase (cat) gene. The cat gene was flanked by two FRT recombination sites for subsequent excision, if desired. This cassette was amplified by PCR to incorporate 30–40 nucleotide overhangs homologous to the yfgG-yfgH intergenic region and introduced into MG1655(pSIM18). Subsequent plasmid removal and Sanger dideoxy sequencing yielded MG1655(yfgG::[Ptrc-[aph Prha-ccdB]FRT-cat-FRT]) (SEQ ID NO:2). This strain was then retransformed with pSIM18 and a PCR product containing a codon-optimized variant of the predicted sequence of Lactobacillus salivarius JCM1046 (GenBank ACL98194.1) and an extension homologous to the ORF arrangement of the Ptrc expression region. pSIM18 was subsequently removed from the strain, and the sequence was confirmed by PCR and Sanger dideoxy sequencing. It was then stored as MG1655 (yfgG::[Ptrc-BSH FRT-cat-FRT]) (SEQ ID NO: 3). BSH gene activity was confirmed by culturing the strain as a single isolate on Lennox LB agar containing CaCl2 and sodium taurodeoxycholate, yielding colonies surrounded by a white halo in the agar. Additionally, thin-layer chromatography of an in vitro sodium taurocholate deconjugation reaction containing cells of the same strain partially lysed by freeze-thaw cycling at -20°C was performed. Cultures of these cells were lysed with bacteriophage P1vir to produce the transducing phage P1vir(yfgG::[Ptrc-BSH FRT-cat-FRT]CmR).

[0133] Transfer of genetic blocks into the M-ACT strain. Using the method described above, strain AZ-39 was isolated from fecal samples of normally developing male wild-type C57B6 mice maintained in the UCSD vivarium. This strain was sensitive to kanamycin, chloramphenicol, carbenicillin, hygromycin, trimethoprim, and ciprofloxacin. The genome sequence of AZ-39 was determined from data acquired from the extracted DNA using a long-read high-throughput sequencing instrument (Pacific Biosciences). AZ-39 did not display any hemolysin or other known toxin genes (e.g., Shiga-like toxin, fragilicin) in its genome sequence.

[0134] AZ-39 was subsequently transduced with P1vir(attB::[aph PrplN-gfpmut2]KmR) to confer kanamycin and chloramphenicol resistance to produce strain AZ-51 (AZ39 GFP+), and then P1vir(yfgG::[Ptrc-BSH FRT-cat-FRT]CmR) to produce strain AZ-52 (AZ-39 GFP+BSH+). Both AZ-51 and AZ-52 fluoresce green under blue light, and when grown as single isolates on solid agar containing sodium taurodeoxycholate, AZ-52 produced halos, whereas AZ-51 did not.

[0135] Colonization of wild-type mice developed normally. Strains AZ-51 and AZ-52 were streaked from their respective glycerol stocks as single isolates. Individual colonies from these streaks were tested for the correct BSH activity phenotype and then inoculated into 1 mL cultures of Lennox LB + 1 mM MgCl2 containing kanamycin (25 μg / mL) for AZ-51 and chloramphenicol (10 μg / mL) for AZ-52. These cultures were grown for 12 hours at 37°C with shaking at 215 RPM. Each culture was then diluted 1000-fold and placed back into 50 mL of the same medium in a 500 mL baffled culture flask supplemented with 1 mM MgCl2 and 0.5× M buffer as described by Studier (2005) Protein Purification & Expression. These cultures were grown for an additional 12 hours at 37°C with shaking at 215 RPM. At the end of the growth period, the absorbance of the culture at 600 nm at 1 cm path length was routinely between 10.0 and 20.0 (taking into account dilution before measurement). Cells were harvested from the measured volume of liquid culture, and the original culture liquid was diluted to 1 × 10 cells. 9 Assuming a cell density of 5 x 10 cells / mL / OD600 10 The cells were resuspended in chilled sterile 1x PBS to a cell density of 1 / mL. This solution was then kept on ice. Within 1 hour, 0.2 mL of this cell suspension was delivered by oral gavage to normally developing wild-type C57B6 mice. The mice were then returned to their enclosures. Cages were changed twice per week for the first week after gavage, then once per week for the next two weeks, and once every two weeks thereafter.

[0136] Fresh fecal samples were collected, homogenized, and plated as described above, except that once homogenized, fecal samples were further plated onto Lennox LB agar containing either kanamycin (25 μg / mL) or chloramphenicol (10 μg / mL) to monitor colonization status. Imaging data were recorded by photographing the resulting plates after growth by blue-light transillumination. Retention of BSH activity was tested by restreaking clones from antibiotic-free plates (e.g., EMB sucrose, MacConkey lactose) onto Lennox LB agar containing CaCl2 and TDCA.

[0137] Example 2 Colonization of the mouse gut with reintroduced and engineered commensal bacteria In this example, a strain of E. coli was isolated from a C57B1 / 6 mouse, engineered to express GFP and bile salt hydrolase genes from Lactobacillus, and then reintroduced into a C57B1 / 6 mouse.

[0138] introduction Dysbiosis is associated with alterations in social, communicative, stress-related, and cognitive behaviors in mouse models (11, 12). Human studies have linked perturbations in the gut microbiome to autism spectrum disorder (13), major depression (14), and Parkinson's disease (12). Growing evidence suggests that microbiome-neuroimmune interactions may mediate the behavioral and physiological abnormalities observed in mouse models, specifically through global changes in the brain transcriptome, altered microglial maturation and function, and blood-brain barrier (BBB) ​​integrity (1, 15). However, it is unclear which agents mediate these effects and through what mechanisms. Here, we review the connections between the gut microbiome, bile acids (BAs), neuroinflammation, and behavioral dysfunction.

[0139] Microbiome and Bile Acid Metabolism: Microbial deconjugation of BAs (i.e., removal of glycine or taurine; Figure 3A) by bile salt hydrolase (BSH) plays an important role in host physiology. Deconjugation prevents BA reuptake from the small intestine, leading to their conversion to secondary BAs in the colon (16). While much of this hydrophobic pool of secondary BAs is excreted, enough is absorbed via passive diffusion to alter serum BA pools and act as signaling molecules (17). BA signaling is mediated by two known receptors: farnesoid X receptor (FXRα) (18) and G protein-coupled BA receptor 1 (TGR5) (19). Metagenomic studies of human populations and mouse models have identified BSH as potentially protective against obesity, metabolic disorders, and other host physiological dysfunctions (20).

[0140] Bile Acids and Neuroinflammation: BAs can regulate neuroinflammation. Both FXRα and TGR5 receptors are found in brain tissues, including microglia and neurons. Ursodeoxycholic acid (UDCA), a secondary BA produced by bacteria, and its hepatic taurine conjugate (TUDCA) are immunomodulatory agents that affect microglia. UDCA inhibits the production of the pro-inflammatory cytokines IL-1β and nitric oxide (NO) and can counteract the effects of neurotoxins on neuronal death and synaptic changes in vitro (21, 22). In mouse models of neuropathology, TUDCA reduced microglial activation, decreased inflammatory cytokines, and preserved neuronal integrity (2, 23). Most studies on BAs and neuroinflammation have used UDCA or its conjugates, but it is unclear whether other BAs have similar effects. The immunomodulatory effects of UDCA are mediated through the TGR5 receptor (5). Indeed, TGR5 agonists also reduced microglial activation and proliferation and proinflammatory cytokines (10). However, other receptors through which BAs can affect neuroinflammation have also been proposed (4).

[0141] High-fat diet, obesity, and neuroinflammation: Consumption of a high-fat diet (HFD) induces neuronal leptin and insulin resistance, disrupting homeostatic signals and creating a positive energy balance (24, 25). Similar to peripheral metabolic tissues (e.g., liver, adipose tissue), neuronal resistance to these signals is associated with the activation of inflammatory signaling cascades (26-28). In the brain, HFD feeding is associated with the expression of pro-inflammatory cytokines, gliosis, altered vasculature, and disrupted BBB permeability (29). Specifically, increased mRNA and protein expression of IL-1β, IL-6, and TNF-α, as well as increased microgliosis, are observed in the hippocampus (29-32).

[0142] High-fat diet and behavioral dysfunction: In rodents, there is ample evidence that HFD-induced neuroinflammation can cause memory dysfunction and anxiety, especially with longer exposure times. Mice fed an HFD (60% kcal from fat) for at least 16 weeks exhibited clear recognition memory impairments (as assessed by a novel object recognition test), whereas mice exposed to an HFD for 5 weeks did not (33-35). Sustained HFD exposure (i.e., >20 weeks) also led to impaired spatial memory and learning, as assessed by the marble burying test, elevated plus maze, and open field tests (29, 31, 36-38), and increased anxiety levels (33, 35, 39). These agnosias were accompanied by neuroinflammation, as determined by increased levels of IL-6 and TNF-α, as well as microglial activation.

[0143] Limitations in Functional Manipulation of the Gut Microbiome: The gut microbiome can regulate various host physiological processes, but how its effects are mediated is unclear. Furthermore, it is unclear whether most interventions targeting microbiome composition (e.g., probiotics, i.e., live bacteria thought to provide health benefits) have a detectable effect on the gut microbiome (40) or whether they are robust to the interpersonal diversity and plasticity of the microbiome in normally developing wild-type (CR-WT) hosts (e.g., humans) (41). To develop a better mechanistic understanding and more effective microbiome-mediated therapies, a different approach focused on functional modulation of the gut microbiome is needed. Current strategies to address this issue have focused on creating engineered bacteria from laboratory-adapted strains. However, these efforts are labor-intensive, costly, and disappointing because these bacteria do not colonize CR-WT hosts (41).

[0144] Herein, we provide methods and compositions to employ "knock-in" functions into the gut microbiome to investigate its effects on luminal ecology, metabolite and nutrient flow, and physiology in CR-WT hosts. While using this tool to investigate the effects of luminal BA biotransformation on host metabolism, we noted that mice in which BSH was knocked in had strikingly clear differences in their behavior.

[0145] Current Paradigm and Its Limitations: New tools are needed to understand whether biochemical functions determined by metagenomics, metabolomics, and / or metatranscriptomics can transmit or disrupt phenotype. Because gut microflora can sense and manipulate the luminal environment, it is becoming an attractive avenue for engineered cell-based therapeutics. However, the inability of engineered laboratory-adapted bacteria to colonize CR-WT hosts in useful numbers and / or for meaningful periods of time and / or to achieve physiological changes in CR-WT mammals has limited their use in mechanistic and therapeutic studies to date (41). Long-term functional manipulation of the gut microbiome of CR-WT hosts with engineered bacteria remains elusive. It is extremely difficult for probiotics (engineered or not) that are not adapted to the host to compete with the microflora already present in the lumen. There are multiple barriers to its survival in the luminal environment, including barriers from the host (e.g., peristalsis, innate and adaptive immunity) and barriers from other natural microorganisms (e.g., competition, availability of niches) ( 42 ).

[0146] It is well recognized that laboratory strains are not suitable vectors for functional delivery. To address this issue, some research groups have developed tools to manipulate bacterial families commonly found in the gastrointestinal microbiome, specifically Bifidobacterium, Lactococcus, and Lactobacillus. By manipulating hundreds of putative commensal species and systematically feeding them to CR-WT mice, these laboratories have searched for long-term colonization. Despite the use of enormous resources, this method has not resulted in colonization in CR-WT hosts. Furthermore, developing colonizing bacteria in this manner would likely require similarly extraordinary resources for each new host (e.g., transgenic mouse, human host) and each new genetic function, making these methods unaffordable for resource-limited laboratories.

[0147] Our new paradigm: We developed a technology that evolved our ability to effectively alter physiological functions in CR-WT hosts using engineered bacteria. We were able to accomplish this by identifying a tractable native / symbiotic bacterium from the CR-WT host, genetically modifying it with genes that confer beneficial functions, and then reintroducing the engineered native bacterium (ENB) into the CR-WT host. Thus, the ENB is already adapted to the luminal environment. Previous resistance to using this method stems from the assumption that native bacteria are difficult to cultivate and modify. We used native E. coli isolated from mouse feces. Bacterial engineering of E. coli can be performed by almost any laboratory with very few resources. Although E. coli is a common native bacterium, many researchers have assumed it to be a poor colonizer due to disappointments they have experienced with laboratory-adapted strains. However, we have demonstrated that (1) After a single gavage, they can colonize hosts maintained on a variety of diets for several months (e.g., stable colonization for at least 140 days), (2) able to express genes (e.g., "knock-in" BSH) that can alter the luminal BA in the gastrointestinal tract; (3) can alter serum metabolites in a predictable manner (e.g., reduce serum conjugated BAs); (4) can alter core metabolic processes of the host; (5) It can change the behavior of animals. They have succeeded in creating engineered bacteria (such as E. coli).

[0148] ENB advances our ability to functionally manipulate the gut microbiome and conduct more mechanistic microbiome studies, such as identifying mediators of the microbiome-gut-brain axis. Specifically, we can determine whether BA deconjugation can affect host behavior and cognition. Furthermore, ENB can be used as a therapeutic agent in CR-WT hosts, such as humans.

[0149] result In vitro metabolite engineering using ENB: Natural microflora provides a rich reservoir of vectors that are easy to handle and can colonize the host. By culturing feces from C57BL / 6 mice, we identified EcAZ, a genetically tractable E. coli strain. We converted EcAZ into ENB expressing BSH by introducing two genes into the chromosome of this strain: a PrplN-GFPmut2 cassette and a Ptrc cassette containing a heterologous gene transfer site in a single step (43). EcAZ was engineered to express the codon-optimized BSH of L. sallivarius (EcAZ). BSH+ ). BSH cleaves taurine from the side chain of BA core sterol (Figure 3A). This new ENB hydrolyzes taurocholate (TCA) to cholic acid (CA) in an in vitro reaction (thin-layer chromatography; Figure 3B). When grown on agar plates containing 10 mM TCA, a white halo of the less soluble hydrolysis product (CA) forms around the BSH-positive strain but not around the parent BSH-negative strain (Figure 3C).

[0150] ENB can colonize the gastrointestinal tract of CR-WT mice after a single gavage: 10-week-old CR-WT C57BL / 6 mice were gavaged with GFP+EcAZ (EcAZ BSH+ / EcAZ BSH- Each mouse was given a single gavage of a stationary-phase culture of GFP+ colony-forming units (CFUs) of GFP+ colonies per gram of stool, approximately 10% over a 20-week period, under both normal chow (NCD) and HFD-fed conditions. 6The colonization of ENB remained stable at 15 weeks (data shown for up to 15 weeks; Figure 4A). The addition of the BSH gene did not affect the ability of ENB to colonize the gastrointestinal tract (Figure 4A). After 8 weeks, some mice were euthanized, and organs were collected and plated to determine the extent of colonization. EcAZ colonized the entire gastrointestinal tract of mice treated with the highest concentration in the terminal ileum and cecum (Figure 4B). No colonization occurred in the spleen, lungs, heart, or liver of the mice. All mice appeared healthy and were indistinguishable from one another. EcAZ BSH+ or EcAZ BSH- Colonization with either EcAZ or EcAZ did not affect body weight (Figure 4C). BSH+ deconjugated BA, but EcAZ BSH- did not deconjugate (Figures 4D, 4E).

[0151] ENB can carry out biotransformation of BAs in the gastrointestinal tract and affect serum BA pools: Fecal analysis showed that the addition of ENB did not significantly shift the microbiome composition (Figure 5A). Targeted metabolomics revealed that EcAZ BSH+ ENB was confirmed to deconjugate primary BAs, which led to metabolomic changes in the luminal BA pool (Figure 5B). Many fecal BAs differed between groups, but the changes were unpredictable. However, changes in serum BA composition were much more predictable. An overall decrease in conjugated BAs was observed (Figure 5C), suggesting that ENB can be used to alter the host's BA composition.

[0152] ENB can induce physiological and behavioral changes: EcAZ BSH+ Metabolic cage assessment of mice revealed a distinct metabolic profile characterized by a low respiratory quotient (RER), suggesting increased fatty acid oxidation (Figure 7A). BA signaling also influences glucose homeostasis; EcAZ BSH+Mice treated with EcAZ had significantly lower postprandial insulin levels (Figure 7B, right). To quantify the observed behavioral differences between mice under these conditions, we performed behavioral and cognitive experiments. BSH+ Mice spent 50% more time on the running wheel compared to the two controls (Figure 7C). BSH+ After 20 weeks, HFD-fed mice performed poorly in this cognitive test, whereas EcAZ-fed mice performed slightly better (Figure 7D). BSH+ Treated HFD mice had no difficulty (Figure 7D). Thus, expression of a single gene in the microbiome, BSH, induces metabolic, behavioral, and possibly cognitive changes in CR-WT hosts.

[0153] Experimental Methods and Design Luminal BA modification by ENB affects cognition in CR-WT mice Our data indicate that high-fat diet (HFD) consumption causes an impairment in novel object recognition. BSH+ HFD mice treated with BAs performed similarly to mice on a normal chow diet (NCD), suggesting that bacterial BA deconjugation affects host cognition.

[0154] Method: ENB colonization and in vivo evaluation. For this experiment, 8-week-old WT male C57BL / 6 mice (72 mice total) were used. Only male mice were used because they are susceptible to diet-induced obesity, whereas female C57BL / 6 mice do not become obese when fed an HFD (44). Body weight and food consumption were monitored for the duration of the experiment. After 2 weeks, 10-week-old mice were treated with PBS, EcAZ, or HCl. GFP+ / BSH- , or EcAZ GFP+ / BSH+Mice are given a single gavage of 100 mg of NCD (24 mice per group). Fecal cultures are used to monitor colonization and BSH activity throughout the experiment. After 2 weeks, half of each group is switched from NCD to HFD (12 mice per condition; LabDiet 58Y1; 18% protein, 61% fat, 21% carbohydrate). After 20 weeks, behavioral testing begins if HFD-related agnosia is observed (34).

[0155] In vivo assessment of luminal and serum BA profiles. After 22 weeks of gavage (20 weeks after dietary change), blood (submandibular) was collected from all mice for targeted BA metabolomics. The effects of ENB on targeted fecal and serum BA pools were assessed by liquid chromatography coupled with mass spectrometry (LC-MS / MS).

[0156] Behavioral testing: The equipment used for behavioral testing is autoclavable and suitable for high-barrier facilities. This is to minimize environmental factors that may affect the composition of the microbiome. In addition, the order of tests is designed to minimize disruption as the test progresses. Milder tests are performed before tests that may cause greater temporary pain or distress, such as tail suspension. Tests are performed in the following order, with 3-5 days between tests:

[0157] Open field test. This is an "emotional" test used to measure anxiety-like responses in rodents exposed to a stressful environmental stimulus (a brightly lit open space) as well as capture measures of spontaneous activity. Each animal is placed in the center of the open arena (45), and several behavioral parameters (distance traveled, speed, time in the center, frequency of time in the center, rearing, and grooming) are recorded and analyzed during a 30-minute observation period.

[0158] Hanging wire test. The hanging wire test allows for the assessment of grip strength and motor coordination (46). Mice are suspended with only their forepaws in contact with an elevated metal bar held parallel to the table by a large ring stand. A score is assigned based on the latency to fall and the hanging strategy.

[0159] Novel Object Recognition Test. This test assays recognition memory while leaving the spatial arrangement of objects intact (47-49). The basic principle is that animals explore a novel environment, but repeated exposure leads to a decrease in exploration (i.e., habituation), and then the novel object is preferentially explored because it is different from what the animal remembers (dishabituation) (50-52). Behavior is videotaped and then scored for contact (nose touching or pointing toward the object within 0.5 cm of the object).

[0160] Marble burying test. The marble burying test is used to assess anxiety-like behavior (53) and obsessive-compulsive behavior (54) by utilizing species-typical burrowing behavior (55). Mice are individually placed in a standard mouse cage containing a 5 cm deep platform with 20 evenly spaced marbles for 30 min, after which the mice are removed and the number of buried marbles (at least 2 / 3 covered by the platform) is determined.

[0161] Barnes maze test for spatial memory. The Barnes maze test is a test of spatial learning and memory, which involves using distal visual cues to avoid a brightly illuminated circular field (56-58). Each session is videotaped, scored by an experimenter blinded to the experimental conditions of the mice, and analyzed to assess distance traveled, speed of movement, and path analysis.

[0162] Tail suspension test. The tail suspension test is a classic test for examining helplessness / depression-like behavior in mice (59, 60). Each mouse is suspended by its tail using adhesive tape on a metal bar placed 30 cm above a flat surface for 6 min. Immobility is quantified by measuring the time during which no whole-body movement is observed. An increase in the time spent immobile is an indicator of increased depressive-like behavior.

[0163] Statistical analysis: StatView (version 5.0.1; SAS Institute Inc.) was used. Data from each behavioral test were evaluated using repeated measures ANOVA with the within-subject factor group and the within-subject factor time or trial, depending on the test. Fisher's protected least significant difference (PLSD) post-hoc test was used when warranted by significant main effects or significant interactions between these effects.

[0164] Necropsy assessment of BA signaling. After behavioral phenotyping, fasting serum and feces were collected from each mouse. Mice were sacrificed using CO2 asphyxiation followed by decapitation. The brain, lungs, heart, liver, spleen, and various segments of the digestive tract were homogenized in PBS, and qPCR was then performed on EcAZ-specific genomic island mRNA (61), GFPmut2, and BSH, as well as cultured tissue, to determine the site of EcAZ colonization. BAs and short-chain fatty acids were measured in serum, feces, liver, brain, and terminal ileum and cecum contents. Host RNA was extracted from the terminal ileum, cecum, liver, and hippocampus. RNA-seq (Illumina HiSeq SE50) was performed on half of the mice (6 per condition). The transcriptome results for key genes were confirmed by qRT-PCR in the other half of the mice (6 per condition).

[0165] Increased bacterial BA deconjugation in the intestinal lumen leads to changes in the BA pool in the terminal ileum, which activates both the FXR and TGR5 pathways. Behavioral tests demonstrated that EcAZ GFP+ / BSH+ Mice, PBS, and EcAZ GFP+ / BSH-The results show that there are cognitive differences between mice from the groups, which could be due to, for example, reduced neuroinflammation, changes in blood-brain barrier permeability, a direct effect of BAs on the neurons themselves, or a combination of factors.

[0166] Luminal BA modification by ENB affects microglial gene expression in CR-WT mice. HFD consumption may cause behavioral dysfunction through multiple mechanisms, including disruption of BBB permeability, increased neuroinflammation, or alterations in the neuronal transcriptome. Because BAs can regulate neuroinflammation, we determined whether ENB affects microglial activation by altering serum BA pools. We note here that BSH activity may induce cognitive changes in the absence of altered microglial activation, and similarly, it may cause changes in microglial activation without inducing cognitive changes.

[0167] Methods: ENB Colonization, In Vivo Assessment, and Necropsy Assessment of BA Signaling. Eight-week-old WT male C57BL / 6 mice (48 mice total) were used. Animals were maintained, assessed, and colonized as described in Specific Aim 1 (SA1), except that eight mice were used in each of the six conditions. These mice were also subjected to all methods used in SA1 to assess colonization and BSH activity.

[0168] Microglial cell isolation. After maintaining mice on an HFD for 24 weeks, microglial cells were isolated as previously described (62). Briefly, after 6 hours of fasting, mice were deeply anesthetized with CO2 and then rapidly perfused intracardially with ice-cold DPBS. The entire brain was removed, and the hippocampus was dissected. One half was used for immunostaining and microglial reconstruction (see below), and the other half was used for microglial isolation and RNA-Seq. The hippocampal tissue was gently homogenized, filtered, and centrifuged. The pelleted homogenate was then resuspended in 37% isotonic Percoll and then overlaid with 70% isotonic Percoll. The tube was then centrifuged, and the 37–70% Percoll interphase was collected and washed with HBSS. The cells were then incubated with CD16 / CD32 blocking antibodies in staining buffer on ice, followed by incubation with anti-mouse CD11b-PE and CD45-Alexa488 antibodies. Sorting was performed and microglia were identified as singlets, CD11b+CD45Low events, which comprised >95% of all CD11b+ events. Isolated microglia were then pelleted and stored at -80°C for downstream protocols.

[0169] RNA isolation, sequencing, and analysis: Total RNA is isolated from microglial homogenates by TRIzol. Library preparation and sequencing are performed by the UCSD sequencing core. Fastq files from RNA-Seq experiments are mapped to individual genomes per mouse strain of origin using STAR.

[0170] Immunostaining and Microglial Reconstruction. Microglial cells were isolated and immunostained using a previously published protocol (12). Dissected hippocampal tissue was fixed in 4% (w / v) paraformaldehyde. 50 mm sagittal sections were created using a vibratome. Free-floating sections were stained with mouse anti-FXR NR1H4, rabbit anti-GPCR TGR5, and goat anti-Iba1, followed by anti-mouse IgG-AF647, anti-rabbit IgG-AF546, and anti-goat IgG-AF488. Sections were mounted and imaged on a confocal microscope. Semi-automated reconstruction of microglial cell bodies and processes was performed. 20–60 cells per animal were analyzed.

[0171] Cytokine quantification. TNF-α, IL-6, and other cytokines are assessed in tissue homogenates and serum using a multiplex platform.

[0172] Increased bacterial BA deconjugation in the intestinal lumen leads to decreased neuroinflammation as determined by morphology (e.g., process diameter, number of branch points, total branch length) and cytokine production (either by multiplex / ELISA or transcription). GFP+ / BSH+ Mice treated with EcAZ had a transcriptome program more similar to NCD control mice, whereas EcAZ GFP+ / BSH- Mice treated with BAs were indistinguishable from the vehicle-treated HFD cohort. Finally, transcriptome pathways will indicate whether the changes induced by increased BA deconjugation are mediated by known BA receptors or some other undefined mechanism.

[0173] By using efficient gut colonizers that bypass the barriers that prevent most probiotics from colonizing the host, ENB has enormous impact on how we analyze and understand the gut microbiome and treat microbiome-mediated diseases. Herein, we provide a basis for using ENB to determine mediators of the microbiome-gut-brain axis. (References) TIFF2023090941000010.tif224169TIFF2023090941000011.tif240169TIFF2023090941000012.tif240169TIFF2023090941000013.tif188169

[0174] Example 3 Colonization of engineered commensal bacteria reintroduced into the human gastrointestinal tract Healthy human subjects provide stool samples on sterile swabs, which are cultured in the laboratory. Probiotic bacterial cells from the stool samples are engineered to express a marker, such as a fluorescent protein. The bacteria transformed with the heterologous polynucleotide are then returned to the subject as a concentrated solution of live organisms, either mixed into food (e.g., oatmeal, yogurt) or as a suspension in a strongly buffered solution packed into large gel capsules immediately before consumption. Human subjects are provided with sterile swabs to collect and submit stool samples at predetermined intervals, e.g., twice a week for two months, to track the success of recolonization of the engineered strain in the subject's digestive tract.

[0175] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and will be encompassed within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19

Claims

1. 1. A substantially homogeneous population of engineered naturally occurring bacterial cells that are probiotic to a mammalian subject for use in a method of treatment of a recipient mammalian subject, comprising: A substantially homogenous population of engineered native bacterial cells is cultured from E. coli bacterial cells isolated from a microbiome sample from a donor subject and transformed to express one or more polynucleotides encoding one or more therapeutic polypeptides and heterologous to the donor subject and / or the bacteria; the engineered naturally occurring bacterial cells have a total in vitro growth or culture time between collection from the donor subject and administration to the recipient mammalian subject of 15 days or less, wherein the in vitro growth or culture time does not include the time the bacterial cells are cryopreserved or lyophilized, but does include the time it takes to transform or introduce one or more heterologous polynucleotides into the bacterial cells; The use comprises administering to a subject at least a portion of the substantially homogeneous, transformed population of isolated and cultured bacterial cells, wherein the administered bacterial cells are capable of establishing in or on the subject for at least two days, resulting in a substantially homogeneous population of engineered native bacterial cells that express one or more therapeutic polypeptides.

2. 2. A substantially homogeneous population of engineered native bacterial cells for use according to claim 1, wherein the cells are autologous to the subject being treated.

3. 3. A substantially homogeneous population of engineered naturally occurring bacterial cells for use as described in claim 1 or 2, wherein the engineered naturally occurring bacterial cells have a total in vitro growth or culture time between collection from the donor subject and administration to the recipient mammalian subject of 14 days or less, or 13 days or less, or 12 days or less, or 11 days or less, or 10 days or less, or 9 days or less, or 8 days or less, or 7 days or less.

4. wherein the one or more polynucleotides encode one or more therapeutic polypeptides and the method of treatment comprises: a) obtaining a microbiome sample comprising bacterial cells from a subject; b) isolating bacterial cells from the microbiome sample, wherein the bacterial cells are derived from a symbiotic / native strain to the subject; c) culturing the isolated bacterial cells in vitro to obtain a substantially homogeneous population of isolated and cultured bacterial cells; and d) transforming a population of bacterial cells with one or more polynucleotides, wherein the one or more polynucleotides are heterologous to the bacteria and / or the recipient.

4. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 3, comprising:

5. 5. The substantially homogeneous population of engineered native bacterial cells for use according to claim 4, further comprising a step of determining and / or measuring the establishment or presence of the administered bacterial cells in or on the recipient mammalian subject.

6. (a) the microbiome sample is obtained from a biological sample selected from the group consisting of bodily excreta, surface biopsies or swabs, and pathology specimens; and / or (b) a detectable portion of the administered bacterial cells stably colonizes the tissue or surface to which it is administered for at least or at least about 3, 4, 5, 6, 7 days; and / or (c) a detectable portion of the administered bacterial cells stably and permanently settles in the tissue or surface to which it is administered; and / or (d) at least or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the administered bacterial cells stably colonize the tissue or surface to which they are administered; and / or (e) the subject is a human; and / or (f) at least or at least about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 and / or (g) the administered bacterial cells are administered to the same tissue or surface from which the microbiome sample was obtained; and / or (h) the administered bacterial cells are administered to the subject multiple times; and / or (i) the administered bacterial cells are administered to the subject at daily, weekly, biweekly, or monthly intervals; and / or (j) the administered transformed bacterial cells do not alter the microbiome of the recipient mammalian subject; 6. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 5.

7. (a) a microbiome sample is obtained from the skin or eye and the administered bacterial cells are administered topically to a subject; or (b) a microbiome sample is obtained from the nasal cavity and the administered bacterial cells are administered via nasal gavage; or (c) a microbiome sample is obtained from the vagina and the administered bacterial cells are administered intravaginally; or (d) a microbiome sample is obtained from the gastrointestinal tract and the administered bacterial cells are administered orally or rectally to the subject; or (e) a microbiome sample is obtained from the gastrointestinal tract and the administered bacterial cells are administered to the subject via a gastric tube or orally in the form of an edible composition; 7. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 6.

8. The edible composition comprises: i) comprises a gel capsule containing the administered bacterial cells or the administered bacterial cells are encapsulated; or ii) selected from the group consisting of yogurt, milk, ice cream, vegetable puree, fruit puree, sherbet, and oatmeal; or iii) is a beverage; 8. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to claim 7.

9. (a) the bacterial cell or population of bacterial cells does not contain a polynucleotide encoding a pathogenic toxin; (b) the bacterial cell or population of bacterial cells is antibiotic sensitive to one or more antibiotic agents used to select the transformed bacterial cells; and / or (c) the bacterial cell or population of bacterial cells is not antibiotic-resistant to one or more clinically used antibiotic agents; 9. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 8.

10. the one or more heterologous polynucleotides encode a fluorescent protein; or the one or more heterologous polynucleotides encode Amuc_1100 of Akkermansia muciniphila, flagellin B of Vibrio vulnificus, elafin, trefoil factor 1 (TFF1), trefoil factor 2 (TFF2), trefoil factor 3 (TFF3), an anti-TNFα antibody / nanobody or a fragment or single chain thereof, cyanovirin-N of Nostoc ellipsosporum, or microcin J25 (MccJ25); 10. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 9.

11. 11. A substantially homogeneous population of engineered native bacterial cells for use according to any one of claims 1 to 10, wherein one or more heterologous polynucleotides encode an enzyme, cytokine or peptide hormone.

12. (a) the enzyme is bile salt hydrolase, N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D, Actinobacillus actinomycetemcomitans dispersin B (DspB), lactase (beta-galactosidase), aldehyde dehydrogenase, alcohol dehydrogenase, bile acid-CoA:amino acid N-acyltransferase (BAAT), phenylalanine hydroxylase, butyrate synthesis pathway enzyme, prolyl endoprotease from Aspergillus niger (AN-PEP), 7-alpha-hydroxysteroid dehydrogenase (7-alpha-HSDH), 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), or cholylglycine hydrolase and cholate 7alpha-dehydroxylase; or (b) the cytokine is selected from mammalian IL-10, mammalian IL-27 dimer (IL27 alpha subunit and / or Epstein-Barr virus-induced 3 (EBI3) subunit expressed separately or as a fusion protein), or TGF-β; or (c) the peptide hormone is selected from the group consisting of mammalian glucagon, glucagon-like peptide 1 (GLP-1), mammalian glucagon-like peptide 2 (GLP-2), fibroblast growth factor 1 (FGF1), fibroblast growth factor 15 (FGF15), fibroblast growth factor 19 (FGF19), insulin, and proinsulin; 12. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to claim 11.

13. (a) the one or more heterologous polynucleotides comprise a codon bias configured to improve or enhance expression of the heterologous protein in the transformed population of isolated and cultured bacterial cells; and / or (b) the one or more heterologous polynucleotides are (i) integrated into the chromosome of the bacterial cells of the transformed population; or (ii) in a plasmid that has been episomally introduced into the bacterial cells of the transformed population; and / or (c) the transformed bacterial cell further comprises a plasmid retention or maintenance system; and / or (d) one or more heterologous polynucleotides are integrated into an expression cassette having at least or at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:2 and are expressed under the control of the Ptrc promoter; and / or (e) the heterologous polynucleotide is expressed under the control of a constitutive promoter, or the heterologous polynucleotide is expressed under the control of an inducible promoter; and / or (f) the population of bacterial cells is freeze-dried or cryopreserved; 13. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 12.

14. The bacterial cells i) capable of metabolizing one or more carbohydrates selected from the group consisting of sucrose, xylose, d-maltose, N-acetyl-d-glucosamine, d-galactose, and d-ribose; ii) utilizes both glycolytic and gluconeogenic substrates; iii) non-motile; iv) capable of producing ribose-5-phosphate; v) able to grow in defined media lacking vitamin B12 (cyanocobalamin); vi) expressing UDP-glucose-4-epimerase and / or glycosyltransferase; vii) contains multiple copies of the gene encoding the β subunit of the tryptophan synthase gene; viii) contains multiple copies of the gene encoding propionate CoA-transferase; ix) expressing capsular polysaccharide (CPS) 4 (CPS4); x) expressing an rnf-like oxidoreductase complex; xi) catabolizing tryptophan to produce indole and other indole metabolites; and / or xii) does not produce any substances that induce double-stranded DNA breaks; 14. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 13.

15. 15. The substantially homogeneous population of engineered native bacterial cells for use according to any one of claims 1 to 14, wherein the one or more heterologous polynucleotides encode an enzyme which is a bile salt hydrolase from the genus Lactobacillus.

16. 16. The substantially homogeneous population of engineered native bacterial cells for use according to claim 15, wherein the bile salt hydrolase is bshA (gene number 3251811) or bshB (gene number 3252955).

17. 17. A substantially homogeneous population of engineered naturally occurring bacterial cells according to any one of claims 1 to 16 for use in the treatment, alleviation, prevention or suppression of obesity, diabetes, cancer such as oral cancer, esophageal cancer, stomach cancer, colon cancer or rectal cancer, ulcerative colitis, Crohn's disease, HIV, a pathogen infection such as Pseudomonas, Clostridium or Salmonella infection, malnutrition, lactose intolerance, phenylketonuria, celiac disease, or brain injury such as traumatic brain injury, neuropathy, dementia, stroke, encephalopathy, hypercholesterolemia, male infertility, female infertility or chronic kidney disease.

18. 18. A substantially homogeneous population of engineered natural bacterial cells for use according to any one of claims 1 to 17, wherein the bacterial cells are derived from a bacterial strain that is native to the recipient subject and / or the bacterial cells are not derived from a laboratory-adapted bacterial strain.

19. the amount of the population of transformed bacterial cells detectable in a sample obtained from the subject does not decrease by more than 10-fold for at least two weeks after administration; and / or The population of transforming bacterial cells detectable in a sample obtained from the subject is 10 CFU / gram for at least 2 weeks after administration. 4 exceeding 19. A substantially homogeneous population of engineered naturally occurring bacterial cells for use according to any one of claims 1 to 18.

Images