Modified bacteria for reproductive health

Abstract

Compositions and methods of treating or preventing a reproductive 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). Compositions and methods for treating or preventing polycystic ovary syndrome (PCOS).

Description

PATENT COOPERATION TREATY APPLICATIONFORMODIFIED BACTERIA FOR REPRODUCTIVE HEALTHCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 677,91 1 , filed July 31 , 2024, which is incorporated herein by reference.TECHNICAL FIELD

[0002] The present invention relates to modification of hormonal conditions that affect reproductive health.BACKGROUND

[0003] Polycystic ovary syndrome (PCOS) is a lifelong multifactorial disorder, affecting up to 18% of reproductive-aged women.1,2It is characterized by androgen excess, ovulatory dysfunction, and polycystic ovaries, and is the leading cause of anovulatory infertility.3’6PCOS independently increases the risks for endometrial hyperplasia and cancer, type II and gestational diabetes, hypertension, non-alcoholic or metabolic dysfunction-associated fatty liver disease, obstructive sleep apnea, anxiety, and depression.3,7The current medical management of PCOS is suboptimal since it focuses on symptoms of the syndrome rather than its root causes. Therapeutic goals for women with PCOS include amelioration of hyperandrogenic features, management of metabolic risk factors, prevention of endometrial hyperplasia, contraception in those not pursuing pregnancy, and ovulation induction in those who are.8However, there are no specific treatments for PCOS. Thus, there is a need for better understanding of the pathophysiology of the disease and development of targeted therapies.

[0004] Importantly, there is a significant relationship between the gut microbiome and PCOS. Individuals with PCOS have gut microbiota communities that are distinct from healthy controls and compositional diversity correlates with clinical parameters.9’12Both human studies and murine models suggest that compositional and functional changes in the microbiome contribute to the underlying mechanisms of PCOS.12’16In the letrozole(LET) model, which uses treatment of an aromatase inhibitor to elevate testosterone levels and recapitulate PCOS pathophysiology, alterations in the gut microbiome are correlated with hyperandrogenism.17-18However, these relationships are not merely correlational. When LET-treated mice were co-housed with littermate controls, the exposure to a healthy gut microbiome ameliorated both the metabolic and reproductive phenotypes of the PCOS mice.13This establishes a direct link between the gut microbiome and PCOS and suggests that microbiome-targeted therapies could be effective for PCOS.

[0005] Bile acids may play a key role in facilitating the connection between the gut microbiome and PCOS. The gut microflora perform a variety of biotransformations to weaken bile acid detergent and antimicrobial properties, primarily by deconjugating them using bile salt hydrolase (BSH) and further modifying them into secondary bile acids,19which act as signaling molecules. Conjugated, deconjugated, and secondary bile acids are versatile, microbially-modified ligands that modulate many physiological processes20including metabolism21-31, neuroinflammation32-36, and circadian rhythms37,38. Bile acids and BSH have been implicated in the pathophy siology of PCOS in multiple human and murine studies.12,39-41However, evidence supporting this link is correlational, inconsistent, and lacks a conclusive mechanism. For example, Qi, et al. showed B. vulgatus, a bacterial species identified in the microbiome of patients with PCOS, was sufficient to recapitulate the reproductive and metabolic effects of PCOS in mice.12The authors showed that effects of B. vulgatus can be reversed with the addition of conjugated bile acids and increased interleukin-22 (IL-22) and thus presumed that its effects were mediated by increased BSH activity. However, the published genomes of B. vulgatus strains show that they contain a gene homologous to BSH that can only deconjugate highly specific moieties of bile acids.42B. vulgatus has extremely limited effects on human bile acids.43The same group demonstrated that removing the bile acid modifying genes from B. vulgatus still contributed to PCOS through a bile acid-independent mechanism involving the metabolite agmatine.44Moreover, women with PCOS have higher levels of conjugated bile acids in systemic circulation suggesting decreased BSH activity.40Thus, though BAs likely contribute to the pathophysiology of the PCOS, the role of bacterial functions in general, and BSH in particular, is not clear. There is a complex interplay between bile acid signaling, insulin resistance, and reproductive features of PCOS. Insulin resistance affects 65-70% of individuals with PCOS, whereas only 40-60% of individuals with PCOS areoverweight or obese, suggesting, along with cellular mechanisms, that insulin resistance is independent of obesity.45'46Bile acids can mediate glucose homeostasis through famesoid X receptor (FXR) or Takeda G-protein-receptor-5 (TGR5).47Further, there is a bidirectional "‘chicken and egg” relationship between insulin resistance and hyperandrogenism in PCOS, each contributing to the maintenance of the other. Insulin sensitizers like metformin can improve serum androgen reduction in conjunction with other therapies, and conversely, antiandrogen therapies can partially reverse peripheral insulin resistance.48Increasing luminal BSH activity improves glucose homeostasis49, therefore microbial BSH could improve reproductive dysfunction in PCOS as a downstream effect of insulin sensitization. BSH and its impact on the bile acid pool could also modulate PCOS reproductive features through the hypothalamic-pituitary-gonadal (HPG) axis directly. Some androgens such as androsterone can activate FXR receptors and influence bile acid synthesis and composition.50Conversely, bile acids can influence plasma testosterone concentrations and alter male fertility in a healthy state and in the context of metabolic syndrome1 52However, whether BAs affect female reproductive hormones and fertility is not known.SUMMARY OF THE INVENTION

[0006] The disclosure provides, in part, methods of treating or preventing a reproductive disease or condition in a mammalian subject. In embodiments, the method comprises 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).

[0007] The disclosure provides a method of modulating reproductive health comprising administering to a subject in need thereof an effective amount of a bacterial cell genetically modified to increase expression of bile salt hydrolase (BSH). In embodiments, the method decreases serum testosterone in the subject, improves glucose homeostasis, body composition, and hormonal dysregulation in a subject. In embodiments, the method is mediated by the bile acid receptors famesoid X receptor (FXR) and Takeda G-protein-receptor-5 (TGR5).

[0008] The disclosure provides a method of treating polycystic ovary syndrome (PCOS) comprising administering to a subject in need thereof an effective amount of abacterial cells genetically modified to increase expression of bile salt hydrolase (BSH). In embodiments, the cells decrease serum levels of testosterone of the subject.

[0009] In embodiments, the genetically modified bacterial 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 enteric cells, such as 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 or gavage.

[0010] In embodiments, the invention provides a method of delivering bile salt hydrolase (BSH) to a mammalian subject in need thereof, the method comprising:

[0011] a) obtaining a microbiome sample comprising bacterial cells from a donating subject;

[0012] b) isolating a bacterial cell from the microbiome sample, wherein the bacterial cell is from a bacterial strain that is commensal / native to the donating subject;

[0013] c) culturing the isolated bacterial cells in vitro to yield a substantially homogeneous population of the isolated and cultured bacterial cells;

[0014] 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

[0015] 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, wherein the administered bacterial cells are capable of colonizing permanently or long-term in or on the mammalian subject and express the BSH.

[0016] 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).BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1I: EcAZ-2BSH+treatment ameliorates metabolic dysfunction in PCOS mice. FIG. 1A, LET Study design and experiment timeline. FIG. IB, ITT after IP injection of insulin. Inset shows area under the curve (AUC). FIG. 1C, Fasted blood glucose levels from ITT timepoint, t = 0. FIG. ID. Daily body weight measurements. FIGS. 1E-F, Fat (e) and lean (f) body mass. FIG. 1G, DHT study design and experiment timeline. FIG. 1H, ITT after IP injection of insulin. Inset shows AUC. FIG. II, OGTT after oral gavage of glucose. Inset shows AUC (b-f, h-i) n = 9-10 mice / group. (c.e,f) Plots show individual points and mean ± SEM. Significance was determined by 2-way ANOVA and is represented by *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001.

[0018] FIGS. 2A-2H: EcAZ-2BSH+treatment doesn’t restore reproductive function in PCOS mice. FIG. 2A, Serum testosterone. FIG. 2B. LH, FIG. 2C, and FSH levels at time of sacrifice. FIG. 2D, Quantitative analysis of % days in diestrus. FIG. 2E, Representative estrous cycles from one mouse per condition. The stages of the estrous cycle are designated as diestrus (D), metestrus (M), estrus (E), and proestrus (P). FIG. 2F, Ovary weight at time of sacrifice. FIG. 2G, Quantitative analysis of corpora lutea (n = 3- 10 / group ) FIG. 2H, Representative histology of ovaries stained with hematoxylin and eosin (H&E) from one mouse per condition. Corpora lutea labeled as (CL). (a-d,f) n = 9- 10 mice / group. (a-d,f-g) Plots show individual points and mean ± SEM. Significance was determined by 2-way ANOVA and is represented by *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.

[0019] FIGS. 3A-3J: Bile acid composition and microbiome composition and function are altered by LET model and EcAZ-2BSH+colonization. FIGS. 3A-D, Total bile acids (a), primary bile acids (b). secondary bile acids (c), and % cholic acid (d) in fecal samples collected from mice prior to sacrifice. Significance was determined by 2-way ANOVA and is represented by *p < 0.05, **p < 0.01, ***p < 0.001. Plots show individual points and mean ± SEM. FIG. 3E, Shannon diversity index (a measure of a-diversity) from metagenomics sequencing of fecal samples collected from mice prior to sacrifice. FIGS. 3F-H, Principal coordinate analysis based on Bray-Curtis dissimilarity (a measure of beta diversity) in all four conditions (f), LET compared to VEH (g), and EcAZ-2BSH+compared to EcAZ-2 in the context of the LET model (h). Significance was determined byPERMANOVA. FIGS. 3I-J, ALDEx2 differential abundance analysis at species level (i) and KEGG orthologue (KO) level (j). Effect size >= 0.5. represents diseaseassociated species and KOs; represents health-associated species and KOs. (e-j) Metagenomic sequencing of fecal samples collected from mice prior to sacrifice, (a-j) n = 9-10 mice / group.

[0020] FIGS 4A-4M: EcAZ-2BSH+effects on PCOS-related metabolic and reproductive phenotype are mediated by FXR and TGR5. LET study in FXR KO (a-f) and TGR5 KO (g-1) mouse cohorts follows the same study design and timeline as the WT LET cohort. FIG. 4A and FIG. 4G, Insulin tolerance test showing blood glucose levels 0-120 minutes after IP injection of insulin. Inset shows AUC. FIG. 4B and FIG. 4H Fasted blood glucose levels from ITT timepoint, t=0. FIG. 4C and FIG. 41 Daily body weight measurements. FIG. 4D and FIG. 4J Lean body mass measured from EchoMRI. FIGS. 4E-4F and FIGS. 4K-4L Serum LH levels (e,k) and ovary weight (f,l) at time of sacrifice. FIG. 4M, Summan' figure of the role of EcAZ-2BSH+and bile acid receptors in PCOS phenotype, (b, d-f, h, j-1) Plots show individual points and mean ± SEM. (a-1) n = 7-8 mice / group. Significance was determined by 2-way ANOVA and is represented by *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.DETAILED DESCRIPTION

[0021] The present disclosure relates, in part, to a method of treating or preventing a reproductive 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).

[0022] In embodiments, the disclosure provides a method of treating polycystic ovary syndrome (PCOS) comprising administering to a subject in need thereof an effective amount of a bacteria genetically modified to increase expression of bile salt hydrolase (BSH). In embodiments, the cells reduce testosterone levels in the subject. 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 cellsare non-native to the subject. In embodiments, the subject is a human. In embodiments, the administration is oral.

[0023] In embodiments, the invention provides that the subject’s enteric bacterial cells can be transformed to express bile salt hydrolase (BSH) in situ, such as through CRISPR engineering.

[0024] In embodiments, the invention provides a method of delivering bile salt hydrolase (BSH) to a mammalian subject in need thereof, the method comprising:

[0025] a) obtaining a microbiome sample comprising bacterial cells from a donating subject;

[0026] b) isolating a bacterial cell from the microbiome sample, wherein the bacterial cell is from a bacterial strain that is commensal / native to the donating subject;

[0027] c) culturing the isolated bacterial cells in vitro to yield a substantially homogeneous population of the isolated and cultured bacterial cells;

[0028] 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

[0029] 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, wherein the administered bacterial cells are capable of colonizing permanently or long-term in or on the mammalian subject and express the BSH.

[0030] In embodiments, the invention provides that the donor subject can be the same as, or different from, the receiving or host subject being treated. 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).

[0031] In embodiments, the invention provides methods of treatment or prevention of a reproductive disease or condition in a mammalian subject, such as treating polycystic ovary syndrome (PCOS). using native bacteria as vectors for the introduction of specific functions into the gut luminal environment to modify host physiology. In embodiments.the invention provides new methods of treatment and prevention to quickly and effectively knock-in a beneficial function in a sustained manner, potentially in perpetuity, by engineering the host’s native microorganisms to induce a physiological change. In embodiments, the invention demonstrates that (1) tractable native bacteria (e.g. E. coll) can be engineered to express a function of interest, (2) these engineered native bacteria, when delivered back to their source host, engraft the entire gut in perpetuity, and (3) they deliver the intended beneficial function and affect host physiology, namely improve insulin sensitivity. Engineered native bacteria can be particularly advantageous at performing bile acid biotransformations, and effecting changes in host metabolism that have implications for host reproductive health.

[0032] 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 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.

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individualpublication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0034] 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.

[0035] 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 Biology7, 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 Enzy mology (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); Cunent 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); Immunobiology7(CA. Janeway and P. Travers, 1997); 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. Harloyv 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 folloyving terms are defined beloyv. Additional definitions are set forth throughout this disclosure.

[0036] 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.

[0037] 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 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.

[0038] 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 amiddle 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.

[0039] 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.

[0040] 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.

[0041] 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 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” canbe used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0042] 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.

[0043] 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.

[0044] 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 mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0045] As used herein, “inactivating mutations” are used to describe mutations, z.e., insertions, deletions, or substitutions, of genomic nucleic acids that result in lack of formation of a transcript or translated product compared to that of a wildtype, or naturally occurring, genomic nucleic acid sequence. An inactivating mutation functionallyinactivates, or renders non -functional and / or inoperative, a naturally occurring nucleic acid sequence for expression.

[0046] As used herein, "‘activating mutations" are used to describe mutations, z.e., insertions, deletions, or substitutions, of genomic nucleic acids that result in increase of formation of a transcript or translated product compared to that of a wildtype, or naturally occurring, genomic nucleic acid sequence. An activating mutation functionally activates, or increases functional operation of a naturally occurring or non-naturally occurring nucleic acid sequence for expression, such as a gene encoding a bile salt hydrolase.

[0047] As used herein, a “knockout” or “KO” is used to refer to genetic manipulation, wherein the manipulation results in a gene being made nugatory and / or the function of the gene is eliminated, either mostly or completely. A knockout may be achieved through various methods known in the art, for example, integration of a premature stop codon or insertions and / or deletions to the degree of rendering the gene inoperative.

[0048] As used herein, a “knockdown” or “KD” is used to refer to genetic manipulation, wherein the manipulation results in a gene’s expression being reduced. A knockdown may be achieved through use of genetic modification resulting in the reduced transcription of a gene or by use of introducing an exogenous polypeptide encoding a short DNA or RNA oligonucleotide(s) that have a sequence complementary to either the gene or an mRNA transcript resulting in lack of abundance of functional gene transcript.

[0049] 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).

[0050] In an aspect, the disclosure provides a purified cell composition comprising one or more of the engineered enteric cell of the disclosure.

[0051] 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%, andmore preferably 80%, 90%, 95%, 98%, 99%, or more of the cells in the composition are of the identified type.

[0052] In some embodiments, at least about 50%. 60%. 70%, 80%, 90%, or 100% of engineered enteric cells express BSH at a higher level than prior to being engineered.

[0053] The disclosure provides methods of making the engineered enteric cells that express BSH.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 know ledgeof the person skilled in the art and may be selected in accordance with the cell the nucleic acid is intended for.

[0058] 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” 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 necessary7elements 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.

[0059] 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 technology7is 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).

[0060] 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, 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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 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.

[0066] 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 (ELIS As and Western blots) or other assays.

[0067] “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.

[0068] “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.

[0069] In an aspect, the disclosure provides a pharmaceutical composition comprising the engineered enteric cell of the disclosure and one or more pharmaceutically acceptable excipients or diluents.

[0070] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises apharmaceutically active agent, such as an engineered bacterial cell or culture or a bile salt hydrolase, 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 earners.

[0071] 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 nonhuman mammals.

[0072] 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 earner. 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.

[0073] 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 bolusadministration 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. Exemplar}’ 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.

[0074] 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 antiinflammatory 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.

[0075] 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 sametime or separately within time intervals. Tn 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.

[0076] 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 reproductive diseases and conditions, such as polycystic ovary syndrome (PCOS).

[0077] 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 entenc cell of the disclosure or the pharmaceutical composition of the disclosure to the subject. In some embodiments, the disease or disorder is polycystic ovary syndrome (PCOS).

[0078] 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 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 methodscontemplated 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.

[0079] In some embodiments, administering comprises administering a therapeutically effective amount to a human subject.

[0080] 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. Tn any case, an effective amount may be given in single or divided doses.

[0081] 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.

[0082] 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 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. Theterms 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."

[0083] 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 w ith 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.

[0084] 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 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.EXAMPLES

[0085] To investigate the specific role of luminal bile acid modifications by BSH on PCOS in vivo, a tool was developed using a native E. coli (EcAZ-2) as a chassis for functional modification of the mouse microbiome.49,53Using an engineered native bacteria that expresses BSH (EcAZ-2BSH+). the inventors facilitated perpetual engraftment into the conventionally raised mouse gut with a single oral gavage. EcAZ-2BSH+functionally alters the bile acid pool without altering overall microbiome composition, and improves glucose homeostasis in wild-type (WT) male and female mice.49Thus, this example assessed whether introducing EcAZ-2DSII )into a mouse model of PCOS could ameliorate disease- associated metabolic and reproductive features or exacerbate it as it has been hypothesized by others.12RESULTS

[0086] ECAZ-2BSH+treatment improves glucose homeostasis and body composition in LET-induced PCOS mice.

[0087] To explore the impact of luminal bile acid modifications in the context of PCOS, we used the aromatase inhibitor, letrozole (LET) to increase androgen levels and recapitulate both the reproductive and metabolic features of PCOS.54Forty conventionally-raised, C57BL / 6 female mice were distributed into four groups based on body weight (n = 10). The groups were colonized with a single gavage of either BSH- containing engineered native E. coli (EcAZ-2BSH+) (n = 20) or a control native E. coli without BSH (EcAZ-2) (n = 20) at 3.5 weeks old. After confirming colonization at -106 CFU / g, half of the mice from each bacterial condition received daily gavage of either LET or vehicle control (VEH). Thus, the study design consisted of 4 groups: VEH EcAZ-2. LET EcAZ-2, VEH EcAZ-2BSH+, and LET EcAZ-2BSH+(n = 10 / group). Towards the endof the 5 week paradigm, we conducted metabolic and reproductive testing to assess PCOS phenotype (Fig. 1A).

[0088] In the EcAZ-2 control mice, LET mice exhibited higher glucose levels during an insulin tolerance test (ITT) with a 30% increase in AUC compared to VEH EcAZ-2 mice (p < 0.001). However, glucose levels were normalized in LET EcAZ-2BSH+mice which demonstrated a 23% reduction in AUC to levels comparable to healthy controls (p < 0.001) (Fig. IB). LET EcAZ-2 mice also had 16% higher fasting blood glucose levels (FBG) compared to VEH EcAZ-2 mice, recapitulating the hyperglycemia in individuals with PCOS (155.6 mg / dL ± 2.9 in VEH EcAZ-2 and 181.0 mg / dL ± 6.9 in LET EcAZ-2; p = 0.0030). ECAZ-2BSH+prevented this LET-induced hyperglycemia with an 11% reduction in FBG (162.9 mg / dL ± 7.2 in LET EcAZ-2BSH+; p = 0.0296) (Fig. 1C). Insulin levels after an overnight fast were elevated in LET EcAZ-2BSH+mice compared to controls, suggesting a compensatory mechanism to restore glucose homeostasis. Overall, these results demonstrate that the microbial bile acid modifications can help correct the glucose homeostasis dysregulation in the letrozole-induced PCOS model.

[0089] Next the effects of our letrozole model on body weight and composition was assessed. LET EcAZ-2 mice had 10% increased body weight at the time of sacrifice compared to VEH EcAZ-2 (17.3 g ± 0.3 in VEH EcAZ-2 and 19.0 g ± 0.4 in LET EcAZ- 2; p = 0.0205). However, LET mice treated with EcAZ-2BSH+did not gain weight (17.5 g ± 0.7 in LET EcAZ-2BSH+) compared to LET EcAZ-2 (Fig. ID). Interestingly, body compositional analysis revealed that the elevated body weight observed in LET EcAZ-2 mice was not due to an increase in adiposity (Fig. IE), corroborated by dissection and measurement of parametrial fat pads after sacrifice. Rather, the increase in body weight was due to a 13% increase in lean body mass in the LET EcAZ-2 condition compared to VEH EcAZ-2 (14.2 g ± 0.3 in VEH EcAZ-2 and 16.0 g ± 0.4 in LET EcAZ-2; p = 0.0042), likely an effect of androgen-mediated muscle hypertrophy. The increase in lean mass was ameliorated in LET EcAZ-2BSH+mice, with a 10% reduction to lean mass and overall body composition similar to controls (14.5 g ± 0.6 in EcAZ-2BSH+LET; p = 0.0173) (Fig. IF). This body weight difference between LET EcAZ-2 and LET EcAZ- 2BSH+occurred in the setting of no decrease in food consumption or caloric intake, but rather a slight increase in LET EcAZ-2BSH+mice (Fig. SID). These results suggest that hyperandrogenic signaling in LET mice may be attenuated with EcAZ-2BSH+colonization.Taken together, these results demonstrate that luminal bile acid deconjugation by BSH ameliorates metabolic features of PCOS in a LET mouse model.ECAZ-2DSII )treatment improves glucose homeostasis in DHT-induced PCOS mice.

[0090] To confirm that this effect wasn’t model specific, the findings were replicated in another mouse model of PCOS using a subcutaneous pellet of the non-aromatizable androgen dihydrotestosterone (DHT). The 90-day DHT model has a stronger dysmetabolic phenotype than the LET model55, and it maintains the core hallmarks of PCOS, including hyperandrogenism, anovulation, and impaired glucose homeostasis. The paradigm for this experiment lasted ninety days to establish both metabolic and reproductive features of PCOS, however, study design and assessments of PCOS phenotype were unchanged (Fig. 1G). After confirming steady colonization with EcAZ-2 and EcAZ-2BSH+, we observed that DHT EcAZ-2 mice had significant impairment in glucose handling compared to VEH EcAZ-2 mice, with a 35% increase in ITT AUC (p < 0.0001). As in the LET model, the DHT ECAZ-2BSH+mice had improved glucose handling, with a 13% reduction in ITT AUC compared to DHT controls (p < 0.05) (Fig. 1H). We also performed an oral glucose tolerance test (OGTT) which similarly demonstrated a prevention of the impaired glucose homeostasis of DHT mice with EcAZ-2BSH+colonization (Fig. II). OGTT was conducted only in the DHT cohort due to the limited time scale of the LET paradigm. In the DHT model of PCOS, we found no significant differences in fasted blood glucose (Fig. IF), fasted insulin, body weight, food intake, adiposity, or lean mass in DHT EcAZ-2BSH+mice compared to DHT EcAZ-2 controls. These results confirm that the beneficial effect of BSH on hyperandrogenic dysmetabolism is observed in multiple models of PCOS, suggesting that bile acids may provide a promising therapeutic target for individuals with PCOS.ECAZ-2BSH+treatment affects some reproductive features of PCOS models.

[0091] Next, whether EcAZ-2BSH+colonization also impacts reproductive features of PCOS was investigated. The LET EcAZ-2 mice recapitulated the hyperandrogenism associated with PCOS, with a %-fold increase in testosterone level compared to VEH EcAZ-2 controls (23.5 ng / dL ± 1.6 inVEH EcAZ-2 and 39.3 ng / dL ± 5.9 in LET EcAZ-2; p = 0.0122) (Fig. 2A). However, EcAz-2BSH+did not prevent hyperandrogenism: serum testosterone levels were similar to those in controls (39.9 ng / dL ± 5. 1 in LET EcAz-2BSH+).The LET model also elevated luteinizing hormone (LH) levels (0.29 ng / mL ± 0.02 in VEH EcAZ-2 and 1.14 ng / mL ± 0.14 in LET EcAZ-2; p < 0.0001), which is elevated in human patients with PCOS. This was partially ameliorated by EcAz-2BSH+treatment (0.83 ng / mL ± 0.10 in LET EcAz-2BSH+, p = 0.0284) (Fig. 2B). Thus, factors other than LH might be maintaining the elevated testosterone levels. Follicle-stimulating hormone (FSH) waselevated in the LET model, but not affected by EcAZ-2BSH+(Fig. 2C). In the DHT model of PCOS, we confirmed that levels of DHT were elevated compared to VEH controls (1303 pg / mL ± 82 in DHT EcAZ-2, 1361 pg / mL ± 133 in DHT EcAz-2BSH+, undetectable in VEH controls, p < 0.0001). In both models, hyperandrogenism in PCOS is driven by exogenous addition of aromatase inhibitor (i.e., LET) or androgen (i.e., DHT). Hence, the primary7cause of hyperandrogenism in both cases cannot be easily reversed by the addition of EcAZ-2BSH+treatment. However, the decrease of LH despite high androgen levels demonstrates that EcAZ-2BSH+impacts one of the key drivers of androgen production that contributes to PCOS symptoms. We next determined whether EcAz-2BSH+treatment affects anovulation and polycystic ovarian morphology (PCOM).ECAZ-2BSH+treatment doesn’t prevent anovulation in PCOS.

[0092] To determine if EcAZ-2BSH+improved anovulation, we conducted vaginal cytology7to determine the estrous cycle of the mice over seven consecutive days. LET mice were stuck in diestrus, indicating anovulation, and cycling was not restored by EcAZ-2BSH+(Fig. 2D-E). Similarly, DHT mice were anovulatory and EcAZ-2BSH+colonized mice remained in diestrus. Ovary weight was increased by 59% in LET EcAZ-2 mice compared to VEH controls, which is associated with hyperandrogenism and PCOM (2.2mg ± 0.2 in VEH EcAZ-2 and 3.5mg ± 0.2 in LET EcAZ-2; p < 0.0001). This increase in ovary weight was ameliorated by 17% by EcAZ-2BSH+(2.9mg ±0.3 in LET EcAZ- 2BSH+; p = 0.0447) (Fig. 2F). To investigate if this reflected PCOM, we conducted H&E staining of ovaries to enumerate the number of corpora lutea (CL), transient structures that emerge following rupture of the developed follicle, indicating ovulation. Histological results confirmed that LET-treated mice were indeed anovulatory, and demonstrated that ECAZ-2BSH+did not rescue PCOM (Figs. 2G-2H). DHT EcAZ-2BSH+mice had no change in ovary weight compared to DHT EcAZ-2 mice, and were anovulatory as detected by ovarian histology7. Taken together, although we observed some subtle reproductivechanges, EcAZ-2BSH+does not prevent acyclicity and anovulation in LET and DHT models of PCOS.LET model and EcAZ-2BSII ttreatment affect fecal bile acid composition.

[0093] It was reported that introduction of EcAZ-2BSH+bacteria to conventionally raised male mice increases the level of fecal total BAs and secondary BAs compared to controls, without changing overall gut microbiome composition in healthy male mice.49We now investigate the effects of luminal BSH overexpression on fecal bile acids and microbiome composition not only in female mice, but also in the context of a PCOS model. Targeted fecal metabolomics revealed that LET decreased total fecal BAs compared to VEH (177.5 ± 28.4 in VEH EcAZ-2 and 42.9 ± 3.6 in LET EcAZ-2; p = 0.0013, Fig. 3A) largely driven by a decrease in primary bile acids (121.0 ± 28.4 in VEH EcAZ-2 and 17.1 ± 4.2 in LET EcAZ-2; p = 0.0052; Fig. 3B). EcAZ-2BSH+did not impact total or primary7bile acid levels in either LET or VEH mice (Figs. 3A,3B). EcAZ-2BSH+did increase levels of secondary BAs in the VEH condition (56.5 ± 8.2 in VEH EcAZ-2 and 92.2 ± 20.2 in VEH EcAZ-2DSH l; p = 0.0328) as we had reported in male mice49, however, it did not have the same effect in the LET condition, perhaps due to the overall reduction in bile acid levels (Fig. 3C). Cholic acid (CA) levels, which were lower in the LET model (7.2 ± 1.4 in VEH EcAZ-2 and 2.6 ± 0.5 in LET EcAZ-2; p = 0.0851), were greater in LET EcAZ-2BSH+mice, at levels similar to healthy controls (8.6 ± 1.7 in LET EcAZ-2BSH+; p = 0.0258) (Fig. 3D). Chenodeoxycholic acid (CDCA) was also significantly increased by EcAZ-2BSH+in LET mice (4.9 ± 0.7 in LET EcAZ-2 and 7.2 ± 0.7 in LET EcAZ-2BSH+; p = 0.0192) but not affected by LET compared to VEH. No other changes were observed in the relative abundance of specific BAs as a percentage of total Bas due to EcAZ-2BSH+. These results indicate that while the LET PCOS model significantly changes the overall bile acid pool, EcAZ-2BSH+more specifically increases the deconjugated primary' Bas CA and CDCA in LET mice.LET model and EcAZ-2BSH+treatment modulate microbiome composition and function.

[0094] To further understand the role of EcAZ-2BSH+on the microbiome, the inventors performed metagenomics sequencing on fecal samples from each condition. Shannon diversity index, which reflects both richness and evenness, revealed a 34% increase in a- diversity in LET mice compared to VEH, and no effect of EcAZ-2BSH+on a-diversity (Fig.3E). Principle coordinate analysis using Bray-Curtis dissimilarity (0-diversity) (Fig. 3F) recapitulated other studies.13,17,18,56in distinguishing microbiome composition between LET and VEH mice (p = 0.018) (Fig. 3G), and as we previously reported in male mice49, ECAZ-2BSH+did not cause overall compositional changes in VEH mice (PERMANOVA p = 0.244) or in LET mice (PERMANOVA p = 0.809) (Fig. 3H). However, LET EcAZ- 2BSH+mice were also indistinguishable from VEH EcAZ-2 (PERMANOVA p = 0.176), suggesting a subtle shift to a microbiome composition between that of LET EcAZ-2 mice and VEH EcAZ-2.

[0095] To determine what compositional changes underlie these differences, the inventors performed differential abundance analysis using ALDEx2 (Fernandes et al., 2014). At the species level, we observed a reduction in the relative abundance of several bacteria within the order Bacteroidales, including the genera Prevotella, Bacteroides, Parabacteroides, and an increase in the relative of abundance of several species within the order Clostridiales, including those from the genus Butyrivibrio, in LET compared to VEH. ECAZ-2BSH+induced unique differences in the relative abundance of species, including a depletion of several bacteria in the genus Lactobacillus. However, the abundance of one species is affected by both LET administration and EcAZ-2BSH+colonization, the archaeon Halobiforma nitratareducens, which is depleted in LET mice compared to VEH but higher in abundance in LET EcAZ-2BSH+compared to LET EcAZ-2 (Fig. 31). To further investigate functional changes, ALDEx2 at the KEGG orthologue (KO) level illustrates a number of functions that follow a similar pattern, more abundant in LET EcAZ-2 compared to VEH controls and less abundant in LET EcAZ-2BSH+compared to EcAZ-2 controls, or vice versa, revealing potential mechanisms through which EcAZ- 2BSH+prevents LET-associated dysbiosis (Fig. 3J). These KOs relate to amino acid metabolism, carbohydrate metabolism, cell growth, and notably, nitrate reduction, an alternative form of respiration that is a capability of H. nitratireducens?7Taken together, these results indicate that EcAZ-2BSH+shifts microbiome function in LET mice to a state more similar to healthy controls.Effects of ECAZ-2BSH+treatment on PCOS metabolic and reproductive features are

[0096] Even with subtle effects on bile acid composition, it was postulated that bile acid receptors mediate the physiological effects of increased luminal BSH activity on PCOS mice. Bile acids are ligands for the nuclear hormone receptor famesoid X receptor (FXR) which is a major metabolic regulator58-60 and may have a direct role in reproductive function.61To investigate the extent to which FXR mediates the effect of EcAZ-2BSH+, we repeated the paradigm outlined in Fig. 2 using Nrlh4- / - mice (FXR KO).

[0097] In the absence of FXR, the engineered native bacteria maintained stable colonization. LET EcAZ-2 mice still demonstrated significant impairment in glucose handling, with a 38% greater ITT AUG compared to VEH EcAZ-2 mice. However, LET ECAZ-2BSH+no longer lowered glucose during ITT or increased fasted insulin levels compared to LET EcAZ-2 in the context of the FXR KO (Fig. 4B. S4B). Similarly, LET treatment in the FXR KO EcAZ-2 mice resulted in a 24% increase in FBG levels (139.43 mg / dL ± 4.87 in VEH EcAZ-2 and 173.0 mg / dL ± 7.4 in LET EcAZ-2; p = 0.0007), which was not prevented by EcAZ-2BSH+in FXR KO mice (Fig. 4B). LET EcAZ-2 mice had 18% greater body weight than VEH EcAZ-2 mice (16.6 ± 0.5 in VEH EcAZ-2 and 18.7 ± 0.2 in LET EcAZ-2; p = 0.0020), which unlike in the WT cohort, was not significantly different between LET mice colonized with EcAZ-2BSH+compared to LET EcAZ-2 controls. (Fig. 4C). There were no significant differences in lean body mass between the four groups (Fig. 4D). As in the WT LET cohort. EcAZ-2BSH+did not affect adiposity' in the absence of FXR. Taken together, these results suggest that global knockout of Nrlh4 didn’t interfere with the LET model’s recapitulation of PCOS dysmetabolism. However. FXR appears to mediate the effect of EcAZ-2BSH+in ameliorating metabolic features of PCOS, suggesting that FXR is necessary' for the beneficial effect of luminal bile acid deconjugation on PCOS pathophysiology.

[0098] With regards to reproductive phenotype in the FXR KO mice, LET EcAZ-2 mice maintained sixfold higher LH levels compared to VEH EcAZ-2 mice (1.48 ng / mL ± 0.3 in VEH EcAZ-2 and 9.4 ng / mL ± 1.4 in LET EcAZ-2; p < 0.0001) (Fig. 2E) and had 34% increased ovary weight (2.06 g ± 0.17 in VEH EcAZ-2 and 2.77 g ± 0.30 in LET EcAZ-2; p = 0.0150) (Fig. 2F). EcAZ-2BSH+mice did not, in the absence of FXR, ameliorate LH levels or ovary' weight in LET mice. As in the WT LET cohort, EcAZ- not afpect serum levels of testosterone and FSH, or prevent anovulation in FXR- / - mice. This suggests that FXR is also not required for the reproductive phenotype of theLET model, but is necessary for the effects of EcAZ-2liSnon LH and ovary weight, revealing that FXR mediates the effect of BSH on both metabolic and reproductive parameters of PCOS.Effects of EcAZ-2BSH+treatment on PCOS metabolic and reproductive features are partially mediated by TGR5.

[0099] Bile acids are also ligands for the G protein-coupled receptor Takeda G- protein-receptor-5 (TGR5), which mediates metabolism62'65and reproduction51’66'68. To test the role of TGR5 in mediating the effect of EcAZ-2BSH+colonization, we repeated the same paradigm (Fig. 1A) again in Gpbarl- / - (TGR5 KO) mice.

[0100] In a global knockout of TGR5, as in the WT LET cohort, EcAZ-2BSH+did not affect colonization levels of the engineered native bacteria. LET mice still demonstrated significantly elevated glucose levels in the ITT compared to VEH controls, with a 38% greater ITT AUC, however LET EcAZ-2BSH+mice did not have improved glucose handling compared to LET EcAZ-2 mice (Fig. 4G). LET EcAZ-2 mice also maintained a significantly higher FBG level compared to VEH EcAZ-2 mice (175.13 mg / dL ± 3.2 in VEH EcAZ-2 and 191 mg / dL ± 6.7 in LET EcAZ-2; p = 0.0204). In contrast to the FXR knockout mice, however, LET EcAZ-2BSH+still demonstrated significantly lower FBG levels compared to LET controls (172.75 mg / dL ± 3.1 in LET EcAZ-2BSH+; p = 0.0086) (Fig. 4H), whereas fasted insulin levels remained unchanged. In the TGR5 KO mice, LET EcAZ-2 mice had significantly greater body weight than VEH EcAZ-2 mice, not prevented by EcAZ-2BSH+(Fig. 41). Similar to the WT LET cohort, in the absence of TGR5, LET EcAZ-2 had an 11% increase in lean mass compared to VEH controls (14.2 ± 0.5 in VEH EcAZ-2 and 15.8 ± 0.4 in LET EcAZ-2; p = 0.0272), which was prevented by EcAZ-2BSH+colonization, with a 10% decrease to lean mass similar to VEH controls (14.4 mg / dL ± 0.4 in LET EcAZ-2BSH+; p = 0.0583) (Fig. 4J). As in the WT LET cohort, EcAZ- 2BSH+not affeC[ adiposity in the TGR5 KO mice. In summary, LET effectively recapitulates the PCOS metabolic phenotype in the absence of TGR5. and TGR5 is necessary for the effect of EcAZ-2BSH+on LET-induced insulin response and body weight, suggesting the effect of bile acid signaling in mediating those features of PCOS are regulated by both FXR and TGR5. TGR5 does not, however, mediate the effect of EcAZ-2BSH+on reducing LET-induced hyperglycemia or lean mass hypertrophy, suggesting that these PCOS features are regulated by FXR alone.

[0101] With regards to reproductive phenotype. LET EcAZ-2 mice had four-fold higher LH levels compared to VEH EcAZ-2 mice in the TGR5 KO (3.6 ng / mL ± 0.8 in VEH EcAZ-2 and 15.3 ng / mL ± 1.4 in LET EcAZ-2; p < 0.0001). Elevated LH levels were ameliorated with a 54% reduction in LET EcAZ-2BSH+mice compared to LET controls (10.0 ng / mL ± 1.4 in LET EcAZ-2BSH+; p = 0.0021) (Fig. 4K). In TGR5 KO mice, serum testosterone levels were nearly doubled in LET EcAZ-2BSH+compared to LET controls (p = 0.0103), so negative feedback from high T levels could explain the decrease in LH level. Similar to WT, LET EcAZ-2 mice had 52.9% greater ovary weight compared to VEH EcAZ-2 mice in the TGR5 KO (2.1 g ± 0.2 in VEH EcAZ-2 and 3.2 g ± 0.2 in LET EcAZ-2: p = 0.0007), however, the increase in ovary weight was not prevented by ECAZ-2BSH+in LET mice (Fig. 4L). As in the WT LET cohort, EcAZ-2BSH+did not impact serum levels of FSH, or prevent anovulation in the absence of TGR5. In addition to the metabolic phenoty pe, LET effectively recapitulates the PCOS reproductive phenotype in the TGR5 KO. and TGR5 is necessary for the effect of EcAZ-2BSH+on LET-induced increase in ovary weight, in addition to FXR. TGR5 is not, however, necessary for the effect of ECAZ-2BSH+in reducing LH levels, suggesting that the effect of bile acid deconjugation on LH levels is mediated more so by FXR. In summary, both FXR and TGR5 play a role in mediating the effect of EcAZ-2BSH+in ameliorating features of PCOS (Fig. 4M).DISCUSSION

[0102] The role of the gut microbiome in PCOS is complex and multidimensional, and the specific role of bile acids on metabolic and reproductive dysfunction has yet to be fully elucidated. In this example, it has been demonstrated that hyperandrogenism, dysregulation of glucose homeostasis, anovulation, and polycystic ovarian morphology are recapitulated by LET and DHT models of PCOS, as seen in previous studies.54,69Engineered native bacteria overexpressing BSH in conventional mice demonstrate that bacterial bile acid biotransformations protect against glucoregulatory dysfunction in PCOS. Importantly, overexpression of BSH did not alter normal female metabolic and reproductive phenotypes in WT mice. Moreover, BSH over-expression improves glucosehandling in two models of PCOS, which both have elevated glucose levels in an ITT but with varying levels of adiposity7, providing evidence for modulation of BSH as a promising therapeutic strategy for both lean and obese individuals with PCOS. LET- treated mice had a shift in the fecal bile acid pool, which has been reported in PCOS patients12,70, where untargeted bile acid metabolomics significantly improves random forest classification accuracy between LET and placebo-treated mice compared to 16S alone.71We replicated a change in the relative abundance of bacteria between LET and control mice that were similar to previous reports, specifically a decrease in species belonging to Bacteroidales and an increase in species belonging to Clostridial es.18Notably, our results did not replicate the higher abundance of B. vulgatus and other Bacteroides species that discriminated PCOS subjects from control subjects in the Qi et al. study.12Analysis of metagenomic sequencing allowed us to further characterize the gut microbiome of LET mice at the level of gene functions, for which we also report differences in the LET model. EcAZ-2BSH+colonization shifts composition and function of the gut microbiome of PCOS model mice, without altering overall microbial diversity7or bile acid levels, demonstrating its efficacy as a precise means to functionally manipulate the gut microbiome.

[0103] ECAZ-2BSH+colonization significantly reduced PCOS-associated elevated LH serum levels and increased ovary weight, demonstrating that luminal BSH over-expression indeed impacts indicators of reproductive function in PCOS. In addition to downstream effects of metabolic improvements, there is evidence that bile acid signaling directly modulates the HPG axis. FXR is present in ovarian follicles73within granulosa cells61, which also contain enzymes for bile acid synthesis76, and bile acids are present in high concentration within bovine and human follicular fluid.7778TGR5 has been identified in ovarian granulosa cells79, and is expressed in the hypothalamus and pituitary of mice80, suggesting potential for direct bile acid receptor activation in the HPG axis. Although it has not yet been investigated in females, FXR and TGR5 activation in testicular Leydig cells regulate testosterone levels and reproductive function in male mice.51’66’67’68Additionally, due to structural similarity between androgens and bile acids50,81-83, crosstalk between these molecules and their receptors are likely to affect reproductive endocrine signaling and PCOS physiology. Neither the FXR or TGR5 KO impaired the estrous cycle of female control mice, suggesting that they are not independently necessary7forreproductive function. Our results suggest that FXR mediates the decrease of LH levels by BSH, and both FXR and TGR5 are necessary' for the effect of BSH on ovary' weight.

[0104] PCOS is a heterogeneous condition whose phenotype varies between individuals based on age, genotype, environment, and lifestyle factors.84There is a growing appreciation for the role of the microbiome in gynecological health85and the reproductive endocrine system.86 87The gut microbiome is increasingly becoming a target for PCOS treatment, with clinical trials demonstrating that pre- and probiotic supplementation reduces testosterone levels and BMI in PCOS patients.88Compositional studies of the microbiomes of PCOS individuals can be biased by dietary' and environmental factors, and methodological variability' between laboratories further contributes to difficulties with replicability and reproducibility.89This underscores the value of mechanistically investigating microbial functions, such as with engineered native bacteria, to identify conserved mechanisms across populations. Since bacterial and bile acid composition was measured in feces, this reflects the microbiome in the large intestine lumen, but not the small intestine where the majority of bile acid biotransformations occur. This caveat could be addressed by a future study characterizing microbial and bile acid composition in ileal contents in response to LET administration and EcAZ-2BSH+- Targeted approaches to unravel metabolic and reproductive features will support tailoring therapeutic approaches to the individual, depending on goals of treatment. Future directions will elucidate the precise pathways through which microbial bile acid deconjugation influences metabolic and reproductive health, in both healthy states and in the context of PCOS. Investigating the interplay between the gut microbiome, insulin signaling, and reproductive endocrine regulation will provide deeper insights into disease mechanisms and lead to potential therapeutics for individuals with PCOS.METHODSANIMAL MODELS

[0105] All animal experiments were conducted in accordance with the guidelines of the IACUC of the University' of California, San Diego. Wild-type female mice (C57BL / 6J) were purchased from Jackson laboratories. FXR- / - and TGR5- / - female mice with a C57BL / 6J background were bred inhouse from adult breeders (STOCK.Nrlh4tmlGonz / J from Jackson laboratories, and obtained from UC Davis-MMRRC. For all studies, mice were housed 2 per cage and pseudorandomized into 4 groups based on initial body weights. All mice were given free access to a normal-chow diet (Diet 7912, Teklad Diets, Madion, WI) and housed in a specific pathogen free barrier facility (irradiated chow and autoclaved bedding).

[0106] For the letrozole-induced PCOS experiments, at 3.5 weeks old, mice in each group were administered a one-time oral gavage of 0.1 ml of 5x1010 CFU / ml of their corresponding bacterial strain. At 4 weeks old, mice in the vehicle groups were orally gavaged daily with 0.5% CMC solution (Sigma-Aldrich), and mice in the letrozole groups were orally gavaged daily with letrozole solution (5mg letrozole / kg bodyweight; dissolved in 0.5% CMC).

[0107] For the DHT-induced PCOS experiments, at 4 weeks old, mice were implanted subcutaneously with either a continuous-release placebo or DHT (Steraloids) implant made with a 12 mm silastic implant (i.d., 1.47mm; o.d., 1.96 mm, Cat# 508-006, Liveo Laboratory Tubing from DuPont, Trademark of the Dow Chemical Company) containing about 8 mg of DHT within a 10 mm space. Implants were made in-house by the Thackray Lab. The pellet release to lasts approximately 4 weeks, so we surgically replaced these pellets with a new pellet 4 weeks after initial implantation.BACTERIAL COLONIZATION

[0108] Bacterial colonization in the mice for the letrozole-induced PCOS experiments was assessed at two timepoints: 3 days after gavage before treatment with letrozole / vehicle solution, and 3-4 weeks after gavage. For the DHT-induced PCOS experiments, we assessed bacterial colonization at 3 timepoints: 1 week after gavage before treatment with DHT / placebo pellet, 3 weeks after gavage, and 6 weeks after gavage. Fecal specimens were collected at ZT 2. Bacterial colonization checks and colonycounts were conducted as previously established in our lab.49ESTROUS CYCLE DETERMINATION

[0109] The stage of estrous cyclicity was determined by vaginal cytology. Vaginal smears from a 20uL saline lavage were obtained from mice over 7-10 consecutive days. To determine estrous cycle, slides were stained using methylene blue dye and scored usinglight microscopic analysis based on visualized cell morphology as described in Byers, et al.90 Proestrus was characterized by the presence of mostly nucleated and some cornified epithelial cells, estrus as mostly cornified cells, and diestrus / metestrus as some cornified epithelial cells and primarily leukocytes.REPRODUCTIVE HORMONE ANALYSES

[0110] Serum testosterone, LH, and FSH were measured using ELISA at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core Facility. Serum DHT was measured using a DHT ELISA kit (Alpha Diagnostic International).METABOLIC PHENOTYPINGOral Glucose Tolerance Test (OGTT)

[0111] For the OGTT, mice were fasted overnight for 14-16 hours. Glucose measurements were taken on a glucometer using one drop of blood from the tail at 0, 30, 60, 90, 120, and 150 minutes after oral gavage of 20% glucose (1g glucose / kg fasted bodyweight). OGTT was conducted only in the DHT cohort due to the limited time scale of the LET paradigm.Insulin Tolerance Test (ITT)

[0112] For the ITT, mice were fasted for 5 hours. Glucose measurements were taken on a glucometer using one drop of blood from the tail at 0, 20, 40, 60, and 120 minutes after oral gavage of insulin, Eli Lilly Humulin R (0.75 - 1.2 IU / kg fasted body weight).Body composition

[0113] Fat and lean mass of live mice was measured using an EchoMRITM 3-in-l Body Composition Analyzer (EchoMRI) with EchoMRI 2022 software.Insulin ELISA

[0114] Insulin was measured using an Ultra Sensitive Mouse Insulin ELISA kit (Crystal Chem) at time of sacrifice after a 14-16hr overnight fast.BLOOD AND TISSUE COLLECTION

[0115] At time of sacrifice, we performed a submandibular cheek bleed or a heart bleed to collect blood. After centrifugation at 1000 g for 20 minutes, we collected serum for hormone measurement assays. Ovaries were collected and fixed in 4% paraformaldehyde (PFA) and stored in 70% ethanol. Zyagen performed paraffin embedding, serial sectioning at 10 micrometers, and hematoxylin and eosin (HE) staining of the ovaries.OVARIAN MORPHOLOGY CLASSIFICATION

[0116] Ovaries were imaged using an Elyra 7 Lattice SIM instrument. Corpora lutea (CL) were identified based on their morphological characteristics, exhibiting luteinization of granulosa cell (GC) layers, and by their continuous visibility across multiple serial sections.91,92For histological analysis, the number of CLs for each ovary were counted. To avoid bias and ensure scientific rigor, corpora lutea were scored by multiple experimenters while blinded to treatment condition.DNA EXTRACTION

[0117] Fresh specimens of mouse fecal samples were collected and flash frozen. All samples were collected at ZT 2 to prevent variability due to time of sample collection.93They were transferred into LOmL Matrix Tubes (MatrixTM LOmL ScrewTop Tubes, 3741, ThermoFisher Scientific, Waltham, MA, USA) containing 95% Ethanol and were delivered to the lab on dry ice for gDNA extraction. Samples then underwent a SpeedVac (Savant™ SpeedVac™ Integrated Vacuum Concentrator Systems, SPD1030-1 15, ThermoFisher Scientific, Waltham, MA, USA) cycle of 45°C for 1 hour to dry residual ethanol. After, a LabTie bead dispenser (Molgen, Veenendaal, Netherlands) was used to dispense 30uL of zirconia-silica beads (BioSpec Products. 0.1, 0.5, 1.0mm Zirconia / Silica Beads, NC0362415, NC0450473, NC9847287, BioSpec, FisherScientific, Hampton, NH, USA) into each sample Matrix Tube. 600uL of lysis buffer (MagMax Microbiome Ultra Lysis Buffer, A42361, ThermoFisher Scientific, Waltham, MA, USA) was also added to each sample tube, then samples were bead beaten for 2 minutes at 1,200 rpm via a SPEXMiniG (HG-400 MiniG® Tissue Homogenizer and Cell Lyser, 1600. SPEX Sample Prep, Metuchen, NJ, USA). After bead beating gDNA extraction proceeded using theMagMAX Microbiome Ultra Nucleic Acid Isolation Kit (A42357, ThermoFisher Scientific, Waltham, MA, USA) on a KingFisher Flex instrument (ThermoFisher Scientific, Waltham, MA, USA), as updated in Shaffer, et al.94METAGENOMIC SEQUENCING

[0118] NovaSeq6000 metagenomic sequencing and iSeq normalization was conducted in accordance with the following protocols, (https: / / doi.org / 10.1186 / sl3059-019-1834-9 (https: / / doi.org / 10. ! 128 / msy stems.00006-23)

[0119] This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S 10 OD026929).

[0120] Raw reads were quality cleaned / trimmed using fastp, then host sequences were removed by using hisat2 to map to the mouse genome (GRCm38.p5), with non-mapping read pair being further processed through kraken2+bracken to drop eukaryotic-mapping reads. Cleaned, hostremoved reads were then mapped to the WoL (v2) database and processed using Woltka to generate taxonomic and functional count tables. Fraction of mapped reads to genomes was a mean of 0.280951 ± std 0.072638. Diversity metrics were calculated using qiime2's “corediversity method, using Chao I alpha diversity and Bray- Curtis beta diversity. Significance was determined by PERMANOVA. Pairwise differential abundance calculations were made using ALDex2 with an effect size <0.05.TARGETED METABOLOMICS

[0121] Bile acids were extracted from samples as described before (https: / / doi.org / 10.1038 / s41467-018-05336-9, https: / / doi.org / 10.1007 / s00216-016-0048- 1). Briefly, stool samples were homogenized and extracted in methanol (10 mg of sample / 100 pL) containing heavy internal standards. Serum samples (25 pL) were extracted with 75 pL of methanol containing heavy internal standards. After vortexing for 10 minutes and centrifuging (16,000 x g, 4 C, 10 min), supernatants were transferred to glass vials for injection. Bile acids were analyzed on a Dionex Ultimate 3000 LC system (Thermo) coupled to a TSQ Quantiva mass spectrometer (Thermo) fitted with a Kinetex C18 reversed phase column (2.6 pm, 150 x 2. 1 mm i.d., Phenomenex). The following LCsolvents were used: solution A, 0.1 % formic acid and 20 mM ammonium acetate in water, solution B, acetonitrile / methanol (3 / 1, v / v) containing 0.1 % formic acid and 20 mM ammonium acetate. The following reversed phase gradient was utilized: at a flow rate of 0.2 mL / min with a gradient consisting of 25-29 % B in 1 min, 29-33 % B in 14 min, 33-70 % B in 15 min, up to 100 % B in 1 min, 100 % B for 9 min and re-equilibrated to 25 % B for 10 min, for a total run time of 50 min. The injection volume for all samples was 10 pL, the column oven temperature was set to 50°C and the autosampler kept at 4°C. MS analyses were performed using electrospray ionization in positive and negative ion modes, with spray voltages of 3.5 and -3 kV. respectively, ion transfer tube temperature of 325°C. and vaporizer temperature of 275°C. Multiple reaction monitoring (MRM) was performed by using mass transitions between specific parent ions into corresponding fragment ions for each analyte. Results were quantified using isotopically labeled internal standards. Data were averaged across samples. The bile acids measured using this technique include CA, TCA, bMCA, TbMCA, a-muricholic acid (aMCA), tauro-a-muricholic acid (TaMCA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), DCA, TDCA, hyocholic acid (HCA), taurohyocholic acid (THCA), m-muri cholic acid (oMCA), tauro-co-muricholic acid (ToMCA), ursodeoxycholic acid (UDCA) and lithocholic acid (LCA). Total bile acid measurements was the sum of all bile acids measured using mass spectrometry. Primary bile acids included CA, TCA, aMCA, TaMCA, bMCA, TbMCA, CDCA, and TCDCA. Secondary7bile acids included DCA, TDCA, oMCA, ToMCA, UDCA, and LCA. Of note, TUDCA was not detected.STATISTICAL ANALYSES

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Shaffer, J.P., Marotz, C., Belda-Ferre, P., Martino, C., Wandro, S., Estaki, M., ... &Knight, R. (2021) A comparison of DNA / RNA extraction protocols for high-throughput sequencing of microbial communities. BioTechniques 70(3). 149-159. https : / / doi. org / 10.2144 / btn-2020-0153.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 disclosed inventive concepts have been described herein in connection with certain embodiments so that aspects thereof may be more fully understood andappreciated, 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.

Claims

1. What is claimed is:

1. A method of modulating reproductive health 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).

2. The method of claim 1, wherein the cells decrease serum testosterone in the subject, improve glucose homeostasis, body composition, and hormonal dysregulation in the subject.

3. The method of claim 1. wherein the disease or condition is polycystic ovary syndrome (PCOS).

4. The method of claim 1. wherein the cells can colonize in the subject permanently or long-term in the mammalian subject and express the BSH.

5. The method of claim 1, wherein the cells colonize in the colon of the subject.

6. The method of claim 1, wherein the cells are Escherichia coli.

7. The method of claim 1, wherein the cells are non-native to the subject.

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 polycystic ovary' syndrome (PCOS) comprising administering or causing to be administered to a receiving mammalian subject an effective amount of at least a portion of a substantially homogeneous and transformed population of isolated and cultured bacterial cells, wherein the administered bacterial cells have been engineered to express bile salt hydrolase (BSH).

11. The method of claim 10, wherein the cells decrease serum testosterone in the subject, improve glucose homeostasis, body composition, and hormonal dysregulation in the subject.

12. The method of claim 10, wherein the disease or condition is polycystic ovary syndrome (PCOS).

13. The method of claim 10, wherein the cells can colonize in the subject permanently or long-term in the mammalian subject and express the BSH.

14. The method of claim 10, wherein the cells colonize in the colon of the subject.

15. The method of claim 10, wherein the cells are Escherichia coli.

16. The method of claim 10, wherein the cells are non-native to the subject.

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

18. The method of claim 10, wherein the administration is oral.

19. An engineered population of enteric bacterial cells, wherein the bacterial cells have been transformed to express bile salt hydrolase (BSH) in amounts effective for the treatment of polycystic ovary syndrome (PCOS).

20. A pharmaceutical composition comprising an engineered population of enteric bacterial cells, wherein the bacterial cells have been transformed to express bile salt hydrolase (BSH) in amounts effective for the treatment of polycystic ovary syndrome (PCOS).

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