Bile salt modification to prevent and treat enteric disease

Abstract

Compositions and methods of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of a bile acid deconjugating agent such as bacteria cells engineered to express bile salt hydrolase (BSH), or an intestinal bile acid receptor agonist such as a TGR5 agonist.

Description

Attorney Docket No.24978-0946 PATENT COOPERATION TREATY APPLICATION FOR BILE SALT MODIFICATION TO PREVENT AND TREAT ENTERIC DISEASE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 582,752 filed September 14, 2023, which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present invention relates to therapies to treat enteric disease and conditions. BACKGROUND

[0003] Ulcerative colitis (UC) is a prevalent inflammatory disease of the colon, and it associated with lower levels of secondary bile acids and bile salt hydrolase (BSH) activity. It has been previously demonstrated that native bacteria can serve as chassis to functionally manipulate the gut microbiome. Intestinal Escherichia coli (E. coli) isolated from mouse feces engineered to express BSH can engraft for years after a single gavage, retain their function, alter luminal and serum metabolome, and affect host physiology without changing the microbiome composition in mice. Other laboratories have demonstrated that secondary bile acids can restrain T helper 17 (Th17) cells and promote regulatory T cell differentiation, thus potentially affect immune response to various stimuli. SUMMARY OF THE INVENTION

[0004] The disclosure provides, in part, compositions and methods of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of a bile acid deconjugating agent. In embodiments, the bile acid deconjugating agent is a live bacterial therapeutic agent (either a single strain probiotic, a microbial community, phage therapy, or engineered bacteria cells) which expresses bile salt hydrolase (BSH). -1- 51630486.4Attorney Docket No.24978-0946

[0005] In embodiments, the agent increases bile salt hydrolase in the gastrointestinal tract of the subject. In embodiments, the agent is a cell engineered to express bile salt hydrolase. In embodiments, the agent reduces inflammatory T cells in the colon of the subject. In embodiments, the enteric disease or condition is inflammatory bowel disease. In embodiments, the enteric disease or condition is Crohn’s disease or ulcerative colitis. In embodiments, the subject is a human. In embodiments, the administration is oral, gastrointestinal or rectal.

[0006] The disclosure provides, in part, compositions and methods of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of an intestinal bile acid receptor agonist. In embodiments, the intestinal bile acid receptor agonist is a TGR5 agonist.

[0007] In embodiments, the agonist is selected from deconjugated bile acid, lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid, cholic acid and oleanolic acid. In embodiments, the agonist is selected from agonists synthesized from C24 bile acids, including 6α-ethyl-23(S)-methylcholic acid (S-EMCA), INT 767 and INT-777, and others provided in Table 1 herein.

[0008] In embodiments, the agonist increases bile salt hydrolase in the gastrointestinal tract of the subject. In embodiments, the agent reduces inflammatory T cells in the colon of the subject. In embodiments, the enteric disease or condition is inflammatory bowel disease. In embodiments, the enteric disease or condition is Crohn’s disease or ulcerative colitis. In embodiments, the subject is a human. In embodiments, the administration is oral, gastrointestinal or rectal. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1E show engineered native bacteria expressing bile salt hydrolase protect from colitis. Engineered native bacteria either expressing (red greyscales line) or not (blue greyscales line) the bile salt hydrolase (BSH) were gavaged into C57BL / 6 males. Colitis was induced by dextran sodium sulfate (DSS) administration (three cycles every two weeks). Fig.1A. Colonization measured as Log(CFU / g of stool) overtime showing that engineered native bacteria colonize the mice gastrointestinal tract for the whole duration of the experiment. Green -2- 51630486.4Attorney Docket No.24978-0946 greyscales rectangles indicate each DSS cycle. Fig. 1B. At the end of the experiment, the colonization by bacteria in the colon was measured as Log(CFU / cm of tissue). BSH+ bacteria (right column) outperformed BSH-bacteria (left column). Fig.1C. Colitis score measured at day 63. Pathologist blindly assessed the tissues for colitis score, including extension of colitis, ulceration, immune infiltrate, loss of tissue architecture. The four degrees of colitis are reported in the table in Fig. 1C. BSH+ bacteria protected mice from severe disease. Figs. 1D-1E. Frequency of CD4 T cells producing IFNg and IFNg / IL-17A double producers measured by flow cytometry in the colon lamina propria.

[0010] Figures 2A-2B show engineered native bacteria expressing bile salt hydrolase protect from weight loss in the T cell transplant model of colitis. Engineered native bacteria either expressing (red greyscales line) or not (blue greyscales line) the bile salt hydrolase (BSH) were gavaged into RAG2KOmales. Colitis was induced by I.P. injection of CD45RBhighCD4 T cells isolated from either WT or TGR5KOdonor mice. Figs. 2A-2B. Weight loss measured over time as % of the initial body weight in RAG2KOmice injected with CD45RBhighCD4 T cells isolated from either WT mice (Fig.2A) or TGR5KOmice (Fig.2B). DETAILED DESCRIPTION

[0011] The disclosure provides, in part, compositions and methods of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of a bile acid deconjugating agent. In embodiments, the bile acid deconjugating agent is a live bacterial therapeutic agent (either a single strain probiotic, a microbial community, phage therapy, or engineered bacteria cells) which expresses bile salt hydrolase (BSH).

[0012] In embodiments, the agent increases bile salt hydrolase in the gastrointestinal tract of the subject. In embodiments, the agent is a cell engineered to express bile salt hydrolase. In embodiments, the agent reduces inflammatory T cells in the colon of the subject. In embodiments, the enteric disease or condition is inflammatory bowel disease. In embodiments, the enteric disease or condition is Crohn’s disease or ulcerative colitis. In embodiments, the subject is a human. In embodiments, the administration is oral, gastrointestinal or rectal. -3- 51630486.4Attorney Docket No.24978-0946

[0013] The disclosure provides, in part, compositions and methods of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of an intestinal bile acid receptor agonist. In embodiments, the intestinal bile acid receptor agonist is a TGR5 agonist.

[0014] In embodiments, the TGR5 agonist is selected from deconjugated bile acid, lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid, cholic acid and oleanolic acid. In embodiments, the agonist is selected from agonists synthesized from C24 bile acids, including 6α-ethyl-23(S)-methylcholic acid (S-EMCA), INT 767 and INT-777, and others provided in Table 1 herein.

[0015] In embodiments, the agonist increases bile salt hydrolase in the gastrointestinal tract of the subject. In embodiments, the agent reduces inflammatory T cells in the colon of the subject. In embodiments, the agent activates intestinal stem cells in the subject. In embodiments, the enteric disease or condition is inflammatory bowel disease. In embodiments, the enteric disease or condition is Crohn’s disease or ulcerative colitis. In embodiments, the subject is a human. In embodiments, the administration is oral, gastrointestinal or rectal.

[0016] The present disclosure relates, in part, to a method of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of a substantially homogeneous and transformed population of enteric bacterial cells, wherein the administered bacterial cells have been engineered to express bile salt hydrolase (BSH).

[0017] In embodiments, the cells reduce colitis and inflammatory T cells in the colon of the subject. In embodiments, the disease or condition is ulcerative colitis. In embodiments, the cells can colonize in the subject permanently or long-term in the mammalian subject and express the BSH. In embodiments, the cells colonize in the colon of the subject. In embodiments, the cells are Escherichia coli. In embodiments, the cells are non-native to the subject. In embodiments, the subject is a human. In embodiments, the administration is oral.

[0018] In embodiments, the invention provides a method of delivering bile salt hydrolase (BSH) to a mammalian subject in need thereof, the method comprising: -4- 51630486.4Attorney Docket No.24978-0946

[0019] a) obtaining a microbiome sample comprising bacterial cells from a donating subject;

[0020] b) isolating a bacterial cell from the microbiome sample, wherein the bacteria cell is from a bacterial strain that is commensal / native to the donating subject;

[0021] c) culturing the isolated bacteria cells in vitro to yield a substantially homogeneous population of the isolated and cultured bacteria cells;

[0022] d) transforming the population of bacterial cells with one or more polynucleotides that are heterologous to the bacteria and / or the donating subject, wherein the one or more polynucleotides encode BSH; and

[0023] 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 bacteria cells, wherein the administered bacteria cells are capable of colonizing permanently or long-term in or on the mammalian subject and express the BSH.

[0024] In embodiments, the invention provides an engineered population of enteric bacterial cells, wherein the bacterial cells have been transformed to express bile salt hydrolase (BSH).

[0025] Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a -5- 51630486.4Attorney Docket No.24978-0946 person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of the compositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.

[0026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0027] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0028] The practice of the present invention may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J .E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993- 1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D .M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F .M. Ausubel et al , eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (J.E. Coligan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA. Janeway and P. Travers, 1997); -6- 51630486.4Attorney Docket No.24978-0946 Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988- 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999). Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0029] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, an engineered enteric cell, a pharmaceutical composition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the engineered enteric cell, pharmaceutical composition and / or method. Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0030] As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consisting of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting -7- 51630486.4Attorney Docket No.24978-0946 of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0031] As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”. It is understood that aspects and embodiments of the invention described herein include “consisting” and / or “consisting essentially of” aspects and embodiments.

[0032] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0033] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e., A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0034] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and / or to “about” another particular value. When -8- 51630486.4Attorney Docket No.24978-0946 such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0035] It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In various embodiments, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0036] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0037] As used herein, “engineered” or “genetically modified” or “transformed” are used interchangeably, wherein a cell has been manipulated by means of molecular reprogramming of a genomic sequence (e.g. by insertion, deletion, or substitution). Said cells include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell and may contain -9- 51630486.4Attorney Docket No.24978-0946 mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0038] The terms “exogenous” and “heterologous” are used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0039] In an aspect, the disclosure provides a purified cell composition comprising one or more of the engineered enteric cells of the disclosure.

[0040] As used herein, a composition containing a “purified cell population” or “purified cell composition” means that at least 30%, 50%, 60%, typically at least 70%, and more preferably 80%, 90%, 95%, 98%, 99%, or more of the cells in the composition are of the identified type.

[0041] In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, or 100% of engineered enteric cells express BSH.

[0042] The disclosure provides methods of making the engineered enteric cells that express BSH.

[0043] Genome editing tools may be used to engineer and / or manipulate cells. In some embodiments, the enteric cell of the disclosure may be engineered with either CRISPR, TALEN, or ZFN genome editing tools.

[0044] Genome editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR) system may be used to genetically modify cells. CRISPR can be used in a wide variety of organisms (e.g., used to add, disrupt, or change the sequence of specific genes). “CRISPR” or “CRISPR gene editing” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas”, as used herein, refers to a CRISPR-associated protein. A “CRISPR / Cas” system refers to a system derived from CRISPR and Cas which can be used to silence, knock out, or mutate a target gene. -10- 51630486.4Attorney Docket No.24978-0946

[0045] The CRISPR / Cas system is based on two elements. The first element is an endonuclease, or Cas, (e.g., Cas9 and MAD7) that has a binding site for the second element, which is the guide polynucleotide (e.g., guide RNA or gRNA). The guide polynucleotide (e.g., guide RNA) directs the Cas protein to double stranded DNA templates based on sequence homology. The Cas protein then cleaves that DNA template. By delivering the Cas protein and appropriate guide polynucleotides (e.g., guide RNAs) into a cell, the organism’s genome is cut at a desired location. Following cleavage of a targeted genomic sequence by a Cas / gRNA complex, one of two alternative DNA repair mechanisms can restore chromosomal integrity: 1) non- homologous end joining (NHEJ) which generates insertions and / or deletions of a few base-pairs (bp) of DNA at the gRNA cut site, or 2) homology-directed repair (HDR) which can correct the lesion via an additional “bridging” DNA template that spans the gRNA cut site. CRISPR / Cas systems are classified by class and by type. Class 2 systems currently represent a single interference protein that is categorized into three distinct types (types II, V and VI). Any class 2 CRISPR / Cas system suitable for gene editing, for example a type II, a type V or a type VI system, is envisaged as within the scope of the instant disclosure. Exemplary Class 2 type II CRISPR systems include Cas9, Csn2 and Cas4. Exemplary Class 2, type V CRISPR systems include, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i and Cas12k (C2c5). Exemplary Class 2 Type VI systems include Cas13, Cas13a (C2c2) Cas13b, Cas13c and Cas13d.

[0046] The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence. As described herein, spacer sequences may also be referred to as “targeting sequences.” In CRISPR / Cas systems for a genetic engineering, the spacers are derived from the target gene sequence (the gNA).

[0047] The targeting sequence can be designed or chosen using computer programs known to persons of ordinary skill in the art. The computer program can use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like. -11- 51630486.4Attorney Docket No.24978-0946 Available computer programs can take as input NCBI gene IDs, official gene symbols, Ensembl Gene IDs, genomic coordinates, or DNA sequences, and create an output file containing sgRNAs targeting the appropriate genomic regions designated as input. The computer program may also provide a summary of statistics and scores indicating on- and off-target binding of the sgRNA for the target gene (Doench et al. Nat Biotechnol.34:184-191 (2016)).

[0048] The target sequence is complementary to, and hybridizes with, the targeting sequence of the gRNA. The target nucleic acid sequence can comprise 20 nucleotides. The target nucleic acid can comprise less than 20 nucleotides. The target nucleic acid can comprise more than 20 nucleotides. The target nucleic acid can comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid can comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

[0049] The CRISPR / Cas system can thus be used to edit a target gene, such as a gene targeted for editing in the cells described herein, by adding or deleting a base pair, introducing a premature stop codon, or introducing a frame-shift mutation which thus decreases expression of the target, in part or completely. The CRISPR / Cas system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to a target gene promoter, sterically blocking RNA polymerases.

[0050] Further aspects of the CRISPR / Cas system known to those of ordinary skill are described in PCT Publication Nos. WO 2017 / 049266 and WO 2017 / 223538, the entire contents of which are hereby incorporated by reference. These and other well-known and new techniques, such as TALEN and Zinc Finger Nucleases, for generating Enteric cells of the present disclosure are contemplated by the present invention.

[0051] In some embodiments, the engineered cells described herein are edited using TALEN gene editing. “TALEN” or “TALEN gene editing” refers to a transcription activator-like effector nuclease, which is an artificial nuclease used to edit a target gene. TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind any desired DNA sequence, including a portion of target genes such as TCR subunits, MHC class I complex -12- 51630486.4Attorney Docket No.24978-0946 components, or CD52. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence, including a target gene sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

[0052] In some embodiments, the engineered cells described herein are edited using ZFN gene editing. “ZFN” or “Zinc Finger Nuclease” or “ZFN gene editing” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit a target gene. Like a TALEN, a ZFN comprises a Fold nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0053] The enteric cells described herein can be modified using methods known in the art. The various gene editing systems described herein may be used to modify the enteric cell to delete, inactivate, reduce expression, or otherwise inhibit function of a target gene or a target gene product.

[0054] The term “nucleic acid” or “polynucleotide”, includes DNA and RNA such as genomic DNA, cDNA and mRNA, or combinations thereof. The nucleic acid may comprise, in addition to the sequence enabling the genetic modifications of the disclosure, further sequences such as those required for the transcription and / or translation of the nucleic acid enabling said genetic modifications. This may include a promoter, enhancer, transcription and / or translation initiation and / or termination sequences, selection markers, sequences protecting or directing the RNA and / or enabling the genetic modifications within the cell. The selection and combination of these sequences is within the knowledge of the person skilled in the art and may be selected in accordance with the cell the nucleic acid is intended for.

[0055] Polynucleotides enabling the genetic modifications of the disclosure may be delivered to cells as an isolated nucleic acid or in a vector. The isolated nucleic acid or the vector may be delivered in lipid- or lipid-based delivery system, such as a liposome. Alternatively, the vector may comprise viral proteins, such as when the vector is a viral vector. The term “vector” -13- 51630486.4Attorney Docket No.24978-0946 as used herein refers to a construction comprised of genetic material designed to direct transformation or transductions of a targeted cell. A vector contains multiple genetic elements positionally and sequentially oriented with other necessary elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary translated in the transfected cells. The term vector as used herein can refer to nucleic acid, e.g., DNA derived from a plasmid, cosmid, phagemid, bacteriophage, virus, retrovirus, adenovirus, adeno-associated virus, lentivirus, or other type of virus into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular proteins. The term “plasmid” as used herein refers to a construction comprised of extrachromosomal genetic material, usually of a circular duplex of DNA which can replicate independently of chromosomal DNA. The plasmid does not necessarily replicate.

[0056] Any suitable vectors are envisaged as within the scope of the instant disclosure. The polynucleotides enabling the genetic modifications of the disclosure can be cloned into a number of types of vectors. For example, the polynucleotides enabling the genetic modifications of the disclosure may be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Expression vectors may be provided to cells, such as enteric cells, in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No.6,326,193).

[0057] The purpose of the vector is to provide a nucleic acid sequence in cells or tissue. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence. Expression products may be proteins, polypeptides, or RNA. The nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous, -14- 51630486.4Attorney Docket No.24978-0946 constitutive, or regulated. The vector can also be used as a prokaryotic element for replication of plasmid in bacteria and selection for maintenance of plasmid in bacteria.

[0058] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0059] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0060] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0061] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0062] In some embodiments, transducing comprises either calcium phosphate-mediated gene transfer, DEAE-dextran-mediated gene transfer, liposome-mediated gene transfer, electroporation-mediated gene transfer, viral vector-mediated gene transfer, or nucleofection- mediated gene transfer. In some embodiments, transducing is accomplished by calcium phosphate-mediated gene transfer. In some embodiments, transducing is accomplished by -15- 51630486.4Attorney Docket No.24978-0946 liposome-mediated gene transfer. In some embodiments, transducing is accomplished by electroporation-mediated gene transfer. In some embodiments, transducing is accomplished by viral vector-mediated gene transfer. In some embodiments, transducing is accomplished by nucleofection-mediated gene transfer.

[0063] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, or confirm effect of genomic modulation, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or other assays.

[0064] “Culture” or “cell culture” refers to the maintenance, growth and / or transformation of cells in an in vitro environment. “Cell culture media,” “culture media” (singular “medium” in each case), “supplement” and “media supplement” refer to nutritive compositions that cultivate cell cultures.

[0065] “Cultivate,” or “maintain,” refers to the sustaining, propagating (growing) and / or transforming of cells outside of tissue or the body, for example in a sterile plastic (or coated plastic) cell culture dish or flask. “Cultivation,” or “maintaining,” may utilize a culture medium as a source of nutrients, hormones and / or other factors helpful to propagate and / or sustain the cells.

[0066] In an aspect, the disclosure provides a pharmaceutical composition comprising the bile acid deconjugating agent, BSH, or an engineered enteric cell expressing BSH, and one or more pharmaceutically acceptable excipients or diluents. In an aspect, the disclosure provides a pharmaceutical composition comprising a live bacterial therapeutic agent (either a single strain probiotic, a microbial community, phage therapy, or engineered bacteria cells) which expresses bile salt hydrolase (BSH), and one or more pharmaceutically acceptable excipients or diluents.

[0067] In another aspect, the disclosure provides pharmaceutical compositions comprising the intestinal bile acid receptor agonist. In embodiments, the intestinal bile acid -16- 51630486.4Attorney Docket No.24978-0946 receptor agonist is a TGR5 agonist. Takeda G-protein coupled receptor 5 (TGR5, also known as GP-BAR1, or M-BAR) is expressed throughout the gastrointestinal tract. In embodiments, the TGR5 agonist is selected from deconjugated bile acid, lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid, cholic acid and oleanolic acid. In embodiments, the TGR5 agonist is selected from agonists synthesized from C24 bile acids, including 6α-ethyl-23(S)- methylcholic acid (S-EMCA), INT 767 and INT-777. TGR5 agonists (or agonists of GP-BAR1 or M-BAR) for use in the present invention are further described in Duboc et al., The bile acid TGR5 membrane receptor: from basic research to clinical application, Digestive and Liver Disease 46: 4, 302-312 (2014) and Zhang et al., Therapeutic opportunities of GPBAR1 in Cholestatic disease, Front. Pharmacol. 12: 805269 (2021), which are incorporated herein by reference in their entireties. A partial list of TGR5 agonists is shown below in Table 1. -17- 51630486.4Attorney Docket No.24978-0946-18- 51630486.4Attorney Docket No.24978-0946

[0068] In an aspect, the disclosure provides a pharmaceutical composition comprising the intestinal bile acid receptor agonist of the disclosure and one or more pharmaceutically acceptable excipients or diluents.

[0069] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.

[0070] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0071] As used herein the term “pharmaceutically acceptable diluent or excipient” or “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which an Enteric cell of the disclosure, is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable diluent or excipient” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated. -19- 51630486.4Attorney Docket No.24978-0946

[0072] Formulations of a pharmaceutical composition suitable for administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable diluents or excipients, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. Formulations may also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents or sterile, pyrogen-free, water. Exemplary administration forms may include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

[0073] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, and / or aromatic substances and the like which do not deleteriously interact with the formulation. In some embodiments, the pharmaceutical composition comprises said Enteric cells in combination with other therapeutically active agents. In some embodiments, the pharmaceutical composition comprises said Enteric cells in combination with antibodies specific to a disease cell phenotype. In some embodiments, the disease cell phenotype is that of a malignant cell. In some embodiments, the disease cell phenotype is that of a viral infection. -20- 51630486.4Attorney Docket No.24978-0946

[0074] The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0075] The present invention provides engineered cells derived from a renewable source that demonstrate BSH expression. These cells provide a promising use for standardized, off-the- shelf cell-based therapies to effectively treat enteric diseases and conditions, such as ulcerative colitis.

[0076] In an aspect, the disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering the engineered enteric cell of the disclosure or the pharmaceutical composition of the disclosure to the subject. In some embodiments, the disease or disorder is ulcerative colitis.

[0077] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their -21- 51630486.4Attorney Docket No.24978-0946 progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0078] In some embodiments, administering comprises administering a therapeutically effective amount to a human subject.

[0079] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a cell to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0080] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0081] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or -22- 51630486.4Attorney Docket No.24978-0946 disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0082] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In some embodiments, the engineered enteric cell or pharmaceutical composition comprising said engineered enteric cell of the disclosure is administered in a prophylactically effective amount.

[0083] In some embodiments, the engineered enteric cell or pharmaceutical composition comprising said enteric cells of the disclosure are administered in combination with a combination partner. The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where the engineered enteric cell, or pharmaceutical composition comprising said engineered enteric cell of the disclosure, and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances the combination partners show a cooperative, e.g., synergistic effect. The terms “co- administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the -23- 51630486.4Attorney Docket No.24978-0946 same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0084] This application incorporates by reference herein the entirety of PCT publication WO2018 / 195097 entitled Engineered Commensal Bacteria and Methods of Use. EXAMPLES

[0085] Methods: A murine-isolated native strain of E. coli was engineered to express a functional BSH enzyme and assessed its effect on disease severity in acute and chronic colitis induced by dextran sulfate sodium (DSS) administration or CD45RBhighCD4 T cells transplant. Body weight as a proxy of animal health / disease status was recorded over time. Colitis severity was assessed histologically after hematoxylin and eosin staining of colonic tissue, and lamina propria T cell analyses by flow cytometry.

[0086] Results: The engineered native E. coli successfully engrafted and colonized the gastrointestinal tract of colitis-afflicted mice after a single gavage and maintained BSH activity throughout the duration of the experiment. Functional expression of BSH resulted in colitis amelioration and protection from severe disease, in both acute and chronic settings. Moreover, fewer inflammatory CD4 T cell populations, including interleukin-17A (IL-17A), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma)-producing T cells, were found in the colonic lamina propria of mice receiving BSH bacteria. BSH-expressing bacteria accumulated mainly in the site of higher colitis activity. Lack of the Takeda G protein-coupled receptor 5 (TGR5) in CD4 T cells results in loss of colitis amelioration. Thus, the therapeutic effect of BSH is mediated by TGR5 receptor expressed on CD4 T cells. -24- 51630486.4Attorney Docket No.24978-0946

[0087] Figures 1A-1E show engineered native bacteria expressing bile salt hydrolase protect from colitis. Engineered native bacteria either expressing (red greyscales line) or not (blue greyscales line) the bile salt hydrolase (BSH) were gavaged into C57BL / 6 males. Colitis was induced by dextran sodium sulfate (DSS) administration (three cycles every two weeks). Fig.1A. Colonization measured as Log(CFU / g of stool) overtime showing that engineered native bacteria colonize the mice gastrointestinal tract for the whole duration of the experiment. Green rectangles indicate each DSS cycle. Fig. 1B. At the end of the experiment, the colonization by bacteria in the colon was measured as Log(CFU / cm of tissue). BSH+ bacteria (right column) outperformed BSH- bacteria (left column). Fig. 1C. Colitis score measured at day 63. The pathologist blindly assessed the tissues for colitis score, including extension of colitis, ulceration, immune infiltrate, loss of tissue architecture. The four degrees of colitis are reported in the table in Fig.1C. BSH+ bacteria protected mice from severe disease. Figs.1D-1E. Frequency of CD4 T cells producing IFNg and IFNg / IL-17A double producers measured by flow cytometry in the colon lamina propria.

[0088] Figures 2A-2B show engineered native bacteria expressing bile salt hydrolase protect from weight loss in the T cell transplant model of colitis. Engineered native bacteria either expressing (red greyscales line) or not (blue greyscales line) the bile salt hydrolase (BSH) were gavaged into RAG2KOmales. Colitis was induced by I.P. injection of CD45RBhighCD4 T cells isolated from either WT or TGR5KOdonor mice. Figs. 2A-2B. Weight loss measured over time as % of the initial body weight in RAG2KOmice injected with CD45RBhighCD4 T cells isolated from either WT mice (Fig.2A) or TGR5KOmice (Fig.2B).

[0089] These findings demonstrate that modulation of bile acids and TGR5 pathway by engineered native bacteria ameliorate colitis in preclinical models and can serve a strategy for functionally curative treatment of ulcerative colitis (UC). Moreover, these results show the potential contribution of bacterial BA biotransformation to the progression and severity of UC.

[0090] It will be understood from the foregoing description that various modifications and changes may be made in the various embodiments of the present disclosure without departing from their true spirit. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. Thus, while the presently -25- 51630486.4Attorney Docket No.24978-0946 disclosed inventive concepts have been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the presently disclosed inventive concepts be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the presently disclosed inventive concepts as defined herein. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the presently disclosed inventive concepts, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments of the presently disclosed inventive concepts only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts. Changes may be made in the construction and formulation of the various components and compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the presently disclosed inventive concepts. -26- 51630486.4

Claims

Attorney Docket No.24978-0946 What is claimed is:

1. A method of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of a bile acid deconjugating agent.

2. The method of claim 1, wherein the agent increases bile salt hydrolase in the gastrointestinal tract of the subject.

3. The method of claim 1, wherein the agent is a cell engineered to express bile salt hydrolase.

4. The method of claim 1, wherein the agent reduces inflammatory T cells in the colon of the subject.

5. The method of claim 1, wherein the enteric disease or condition is inflammatory bowel disease.

6. The method of claim 1, wherein the enteric disease or condition is Crohn’s disease.

7. The method of claim 1, wherein the enteric disease or condition is ulcerative colitis.

8. The method of claim 1, wherein the subject is a human.

9. The method of claim 1, wherein the administration is oral.

10. A method of treating or preventing an enteric disease or condition in a mammalian subject comprising administering to a mammalian subject in need thereof an effective amount of an intestinal bile acid receptor agonist.

11. The method of claim 10, wherein the bile acid receptor is TGR5. -27- 51630486.4Attorney Docket No.24978-0946 12. The method of claim 11, wherein the agonist is selected from deconjugated bile acid, lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid, cholic acid and oleanolic acid.

13. The method of claim 11, wherein the agonist is selected from agonists synthesized from C24 bile acids, including 6α-ethyl-23(S)-methylcholic acid (S-EMCA), INT 767 and INT-777.

14. The method of claim 11, wherein the agonist reduces inflammatory T cells in the colon of the subject.

15. The method of claim 10, wherein the enteric disease or condition is inflammatory bowel disease.

16. The method of claim 10, wherein the enteric disease or condition is Crohn’s disease.

17. The method of claim 10, wherein the enteric disease or condition is ulcerative colitis.

18. The method of claim 10, wherein the subject is a human.

19. The method of claim 10, wherein the administration is oral. -28- 51630486.4

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