Compositions and methods for microbiome regulation

Abstract

To provide compositions and methods for microbiome regulation. [Solution] This disclosure provides techniques for modulating the microbiome of mammalian subjects (e.g., human subjects). This disclosure, in particular, provides therapeutic compositions and methods of using the same, the therapeutic compositions comprising an engineered population of therapeutic bacteria that (i) are nonpathogenic and symbiotic in the subject to be administered and (ii) are resistant to one or more target bacteriophages. In some embodiments, such therapeutic compositions may be useful in treating subjects suffering from or susceptible to diseases, disorders, or conditions associated with microbiome dysfunction (e.g., inflammatory bowel disease).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 62 / 902,327, filed on 18 September 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Recent studies have demonstrated that the mammalian digestive tract harbors a diverse array of beneficial symbiotic bacteria. Reduced abundance and diversity of symbiotic bacteria in the human microbiome are associated with a wide range of diseases, including inflammatory bowel disease, metabolic disorders, allergies, asthma, and autism spectrum disorders. Animal studies have further validated this, showing that transplantation of disease-associated microbiomes into healthy mice can induce disease phenotypes. (Garrett, W. Set al. Cell 131, 33-45 (2007), Ellekilde, M. et al. Sci Rep 4, 5922 (2014), Sharon, G. et al. Cell 177, 1600-1618.e17 (2019)). In recent decades, significant effort and resources have been dedicated to developing bacterial therapies to treat diseases associated with dysbiosis of the gut microbiome. These therapies generally aim to replenish the beneficial bacterial populations that are found to be diminished in disease states. However, most probiotics have shown limited benefits in the treatment of chronic diseases, and systematic reviews have not reported on the effects of probiotics on fecal microbiota composition. Kristensen, NB et al. Genome Med 8, 52 (2016). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Garrett,WSet al.Cell 131,33-45(2007) [Non-Patent Document 2] Ellekilde,M.et al.Sci Rep 4,5922(2014) [Non-Patent Document 3] Sharon,G.et al.Cell 177,1600-1618.e17(2019) [Non-Patent Document 4] Kristensen,NBet al.Genome Med 8,52(2016) [Overview of the project] [Means for solving the problem]

[0004] While much of the focus has been on the bacterial components of the microbiome, bacteriophages—viruses that infect bacteria—constitute at least half of the organisms in the microbiome. Existing research may suggest an increase in viral populations in certain disease states, but the bacterial targets of disease-associated viruses remain unknown, and the effects of bacteriophages present in the human gut, for example, on beneficial bacteria in the human gut, have not yet been characterized. Furthermore, a general lack of predatory interactions between phages and bacteria in the human gut has been proposed. Reyes, A. et al. Nature 466, 334-338 (2010), Chehoud, C. et al. MBio 7, e00322 (2016). This disclosure provides, in particular, insight into predatory interactions between bacteriophages and bacteria in certain human body sites, in contrast to known views in the art. Such insight is partly based on the present invention that bacteriophages present in the human gut can deplete populations of healthy and beneficial bacteria. In particular, the inventors have found that the addition of a viral fraction containing bacteriophages concentrated from fecal samples of individuals (e.g., in some embodiments, patients with diseases associated with microbiome dysfunction, such as inflammatory bowel disease (IBD)) similarly depletes beneficial bacterial species concentrated from samples of the same individuals.

[0005] In particular, this disclosure also provides the insight that bacteriophages that infect Clostridia bacteria are significantly more abundant in patients with IBD, a microbiome-associated disease. Predatory interactions between phages and beneficial bacteria have not yet been investigated in the context of disease. The finding that phages that attack beneficial bacteria are present in patients with IBD provides the insight that the abundance or presence of such phages may contribute to or promote the reduction of beneficial bacteria, such as Clostridia bacteria, in patients with microbiome-associated diseases, such as IBD. Thus, this disclosure further provides the insight that administration of beneficial bacteria is not necessarily effective in treating microbiome-associated diseases because the presence of bacteriophages in patients with microbiome-associated diseases can deplete such administered bacteria.

[0006] The technologies presented herein address one or more sources of problems associated with certain conventional approaches to treating diseases related to microbiome dysfunction based on the administration of beneficial bacteria. For example, the present invention provides, among other things, therapeutic compositions comprising or containing phage-resistant nonpathogenic symbiotic bacteria, and various methods and / or materials related thereto, including, for example, methods of administering such compositions to treat diseases or disorders related to microbiome dysfunction.

[0007] In some embodiments, a method is provided which involves exposing an object suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction to a population of therapeutic bacteria that is (i) nonpathogenic and symbiotic in the object and (ii) resistant to one or more bacteriophages.

[0008] In some embodiments, the therapeutic bacteria are exposed to a subject that needs it (e.g., a subject suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction), and each contains clustered regularly interspaced short palindromic repeats (CRISPR) spacers that target one or more bacteriophages. In some embodiments, the therapeutic bacteria are exposed to a subject that needs it (e.g., a subject suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction), and each contains at least one or more variants of bacteriophage receptor(s) on the bacterial cell surface.

[0009] In some embodiments, a population of such therapeutic bacteria is exposed to a subject suffering from or susceptible to a disease, disorder, or condition associated with gut microbiome dysfunction. Exemplary diseases, disorders, or conditions associated with gut microbiome dysfunction include, but are not limited to, inflammatory bowel disease (IBD) or irritable bowel syndrome, Crohn's disease, ulcerative colitis, immunotherapy-related colitis. In some such embodiments, the therapeutic bacteria exposed to a subject suffering from or susceptible to a disease, disorder, or condition associated with gut microbiome dysfunction can be Caudovirales or resistant to one or more bacteriophages that can include it.

[0010] In some embodiments, the step of exposing a subject that needs it to a population of therapeutic bacteria (e.g., those described herein) includes administering a composition comprising the population of therapeutic bacteria (e.g., those described herein) to such a subject.

[0011] In some embodiments, exposing a subject that needs it to a population of therapeutic bacteria (e.g., those described herein) involves administering to the subject a composition comprising a nucleic acid sequence for altering the genome of host commensal bacteria such that the host commensal bacteria are genetically engineered to be resistant to a target bacteriophage. Such a composition is delivered to the host commensal bacteria in the subject that needs it to produce the therapeutic bacteria described herein. Methods for delivering a composition comprising a nucleic acid sequence are known in the art, and those skilled in the art will understand that in some embodiments, such nucleic acid sequences may be delivered by recombinant bacteriophage, and in some embodiments, such nucleic acid sequences may be delivered by a vector. In some embodiments, the nucleic acid sequence for altering the genome of host commensal bacteria comprises one or more CRISPR spacers targeting one or more target bacteriophages.

[0012] In some embodiments comprising the therapeutic bacteria described herein, the bacteriophages to which such therapeutic bacteria are resistant are associated with diseases associated with microbiome dysfunction. In some embodiments, the bacteriophages to which such therapeutic bacteria are resistant are temperate or non-lytic bacteriophages.

[0013] In some embodiments of the therapeutic bacteria described herein, such a population of therapeutic bacteria comprises at least one isolated, purified, or cultured symbiotic bacteria (e.g., including at least two, at least three, at least four, at least five, or more) selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Coprococcus, Clostridium, Collinsella, Desulfomonas, Dorea, Escherichia, Eubacterium, Fusobacterium, Gemmiger, Lactobacillus, Lactoccucs, Monilia, Peptostreptococcus, Propionibacterium, Ruminococcus, and combinations thereof. In some embodiments, the population of therapeutic bacteria comprises Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, Lactoccucs, or combinations thereof. In some embodiments, such bacteria may be self-derived. In some embodiments, such bacteria may be homogeneous.

[0014] Another aspect described herein relates to a therapeutic composition comprising an engineered population of therapeutic bacteria that (i) is nonpathogenic and symbiotic in the target to be administered, and (ii) is resistant to one or more target bacteriophages.

[0015] In some embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein each contain one or more CRISPR spacers that target one or more target bacteriophages. In some such embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein are genetically engineered to express one or more CRISPR spacers that target one or more target bacteriophages.

[0016] In some embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein each comprise at least one variant of a bacteriophage receptor(s) on the bacterial cell surface. In some such embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein are genetically engineered to express at least one variant of a bacteriophage receptor(s) on the bacterial cell surface.

[0017] In some embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein include at least one isolated, purified, or cultured bacteria (e.g., at least two, at least three, at least four, at least five, or more) selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Coprococcus, Clostridium, Collinsella, Desulfomonas, Dorea, Escherichia, Eubacterium, Fusobacterium, Gemmiger, Lactobacillus, Lactoccucs, Monilia, Peptostreptococcus, Propionibacterium, Ruminococcus, and combinations thereof. In some embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein include Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, Lactoccucs, or combinations thereof. In some embodiments, such bacteria may be autologous. In some embodiments, such bacteria may be homogeneous.

[0018] The technologies provided herein may be useful for the treatment and / or prevention of diseases, disorders, or conditions associated with microbiome dysfunction. Therefore, the technologies provided herein are suitable for subjects suffering from or susceptible to diseases, disorders, or conditions associated with microbiome dysfunction. In some embodiments, the technologies provided herein are suitable for subjects suffering from or susceptible to diseases, disorders, or conditions associated with intestinal microbiome dysfunction. Examples of diseases, disorders, or conditions associated with intestinal microbiome dysfunction include, but are not limited to, inflammatory bowel disease (IBD) or irritable bowel syndrome, Crohn's disease, ulcerative colitis, and immunotherapy-associated colitis. In some embodiments, subjects receiving the therapeutic bacteria described herein may have previously undergone probiotic therapy, fecal microbiota transplantation (FMT), and / or immunotherapy (e.g., colitis-associated immunotherapy).

[0019] These and other embodiments included in this disclosure are described in more detail below and in the claims. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method comprising the step of exposing an object suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction to a population of therapeutic bacteria that is (i) nonpathogenic and symbiotic in the object and (ii) resistant to one or more target bacteriophages. (Item 2) The method according to item 1, wherein the therapeutic bacteria each include at least one clustered, regularly spaced, short-interval palindrome repeat (CRISPR) spacer, each targeting one or more target bacteriophages. (Item 3) The method according to item 1 or 2, wherein each therapeutic bacterium comprises a mutation in one or more receptors of the therapeutic bacterium for a target bacteriophage receptor-binding protein. (Item 4) The method according to item 2 or 3, wherein the microbiome is the gut microbiome. (Item 5) The method according to item 4, wherein the disease, disorder, or condition associated with the intestinal microbiome dysfunction is inflammatory bowel disease (IBD) or irritable bowel syndrome. (Item 6) The method according to item 4, wherein the disease, disorder, or condition associated with the aforementioned intestinal microbiome dysfunction is Crohn's disease. (Item 7) The method according to item 4, wherein the disease, disorder, or condition associated with the aforementioned intestinal microbiome dysfunction is ulcerative colitis. (Item 8) The method according to item 4, wherein the disease, disorder, or condition associated with the aforementioned intestinal microbiome dysfunction is immunotherapy-associated colitis. (Item 9) The one or more target bacteriophages are associated with the microbiome dysfunction. The method described in any one of items 1-8, which is associated with the disease. (Item 10) The method according to any one of items 1 to 9, wherein the one or more bacteriophages are temperates or insoluble bacteriophages. (Item 11) The method according to any one of items 1 to 10, wherein the one or more bacteriophages are Caudovirales or include them. (Item 12) The method according to any one of items 1 to 11, wherein the therapeutic bacteria are Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, Lactoccucs, or a combination thereof, or include them. (Item 13) The method according to any one of items 1 to 12, wherein the subject is receiving probiotic therapy, fecal microbiota transplantation (FMT), and / or immunotherapy (e.g., colitis-related immunotherapy). (Item 14) The method according to any one of items 1 to 13, wherein the exposure step comprises administering to the subject a composition comprising the population of the therapeutic bacteria. (Item 15) The method according to any one of items 1 to 14, wherein the exposure step comprises administering to the subject a composition comprising a nucleic acid sequence encoding the CRISPR spacer, the composition being delivered to host symbiotic bacteria of the subject to produce a therapeutic microorganism. (Item 16) The method according to item 15, wherein the nucleic acid is delivered by a recombinant bacteriophage. (Item 17) The method according to item 15, wherein the nucleic acid is delivered by a vector. (Item 18) A therapeutic composition comprising an engineered population of therapeutic bacteria that (i) is nonpathogenic and symbiotic in the target to which it is administered, and (ii) is resistant to one or more target bacteriophages. (Item 19) The therapeutic composition according to item 18, wherein the therapeutic bacteria comprises clustered, regularly spaced, short-interval palindromic repeat (CRISPR) spacers, each targeting one or more target bacteriophages. (Item 20) The therapeutic composition according to item 18, wherein the therapeutic bacteria are genetically engineered to express a CRISPR spacer that targets one or more target bacteriophages. (Item 21) The therapeutic composition according to any one of items 18 to 20, wherein the therapeutic bacteria are Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, Lactococcus, Akkermansia, or a combination thereof, or comprising them. (Item 22) A therapeutic composition according to any one of items 18 to 21, wherein one or more receptors of the therapeutic bacterium for a target bacteriophage receptor-binding protein are mutated such that the therapeutic bacterium is resistant to the target bacteriophage. [Brief explanation of the drawing]

[0020] [Figure 1] This is a schematic diagram illustrating an exemplary in vitro assay for bacteriophage and bacterial host identification. A shows the isolation of bacteria from patients with inflammatory bowel disease (IBD) or healthy patients for the preparation of bacterial cultures. B shows the addition of isolated virus-like particles (VLPs) from the same patients to the bacterial cultures in the presence of mitomycin C (an active agent that induces temperate phage induction). E. coli and T7 phage were added to subsets of the cultures as positive controls. The cultures were grown anaerobically for 72 hours. The bacterial and viral fractions of the cultures were then separated for 16s sequencing. [Figure 2A] This graph shows the predatory interactions between bacteriophages and bacteria in the human gut. It shows the abundance of bacterial populations determined from 16s sequencing. VLP11-15 refers to a group of samples containing bacterial compositions without phage addition. VLP21-25 refers to a group of samples containing bacterial compositions from cultures with phage addition. Phage addition causes a reduction in Clostridium scindens and Bifidobacterium longum species. VLP31-35 refers to a group of samples containing bacterial compositions without phage, with mitomycin C added. VLP41-45 refers to a group of samples containing bacterial compositions with both phage and mitomycin C added. Mitomycin C addition causes a significant change in the bacterial community, indicating the induction of prophages that attack these bacteria. VLP51-55 refers to a group of samples containing bacterial communities spiked in with E. coli. VLP61-65 refers to a group of samples containing bacterial communities spiked in with E. coli and T7 phage. E. coli was eliminated in the presence of T7 phage, indicating the validity of the assay. [Figure 2B]This graph shows the predatory interactions between bacteriophages and bacteria in the human gut. It indicates the presence of gut phages that deplete the following "beneficial" gut bacteria: Clostridium scindens. [Figure 2C] This graph shows the predatory interactions between bacteriophages and bacteria in the human gut. It indicates the presence of the following gut phage that depletes "beneficial" gut bacteria: Bifidobacterium longum. [Figure 3A] This is a schematic diagram illustrating an exemplary computational approach for identifying bacteriophages and bacterial hosts. The viral sequence is matched to a CRISPR spacer from a known bacterial host to identify the putative bacterial host. [Figure 3B] This is a schematic diagram illustrating an exemplary computational approach for identifying bacteriophages and bacterial hosts. The viral sequence is matched to a CRISPR spacer from a known bacterial host to identify the putative bacterial host. [Figure 4] This graph shows the phage populations that infect Clostridia bacteria, which are more abundant in patients with inflammatory bowel disease (IBD). The phage sequences present in individuals with IBD were cross-referenced against a curated collection of CRISPR spacer sequences found within the range of intestinal bacteria. [Figure 5] This graph shows the presence of phage populations in the intestines of patients with inflammatory bowel disease (IBD) that infect various strains of Clostridia. The phage sequences present in individuals with IBD were cross-referenced against a curated collection of CRISPR spacer sequences found within the range of intestinal bacteria. [Modes for carrying out the invention]

[0021] Specific definition Administration: As used herein, the terms “administer” or “dosage” typically refer to the administration of a composition to a subject to achieve delivery of an active substance to that subject. In some embodiments, the active substance is a composition or is contained in a composition. Those skilled in the art will recognize the various routes that may be used to administer to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In many embodiments provided by this disclosure, administration is oral administration. In some embodiments, administration may consist of only a single dose. In some embodiments, administration may consist of the application of a number of doses. In some embodiments, administration may consist of intermittent (e.g., multiple doses separated by time intervals) dosing and / or periodic (e.g., individual doses separated by time intervals) dosing. In some embodiments, administration may consist of continuous dosing (e.g., perfusion) over at least a selected period of time. Cell administration may be carried out by any suitable route that results in delivery of at least a portion of the cells or cellular components to be delivered to a desired location within the subject in which viable cells can be delivered.

[0022] Related: The term “associated” is used herein to describe two events or entities when the presence, level, and / or form of one correlate with that of the other. For example, a particular entity (e.g., a bacteriophage) or a particular event (e.g., a microbiome dysfunction) is considered associated with a disease, disorder, or condition if its presence, level, and / or activity correlate with the incidence or susceptibility to that disease, disorder, or condition.

[0023] Bacteriophage: As used herein, the term “bacteriophage,” synonymous with the term “phage,” has its conventional meaning as understood in the art, namely, a virus that infects or selectively infects prokaryotes such as bacteria and replicates within them. Bacteriophages include wild-type, naturally occurring, isolated, or recombinant bacteriophages. In some embodiments, bacteriophages are specific to a particular genus or species or strain of bacteria.

[0024] Bacteriophage-resistant bacteria: As used herein, the term “bacteriophage-resistant” refers to a bacterial strain that is partially or completely resistant to one or more bacteriophages. Partially resistant strains are those that do not always defend against or inhibit phage infection. For example, a partially resistant strain is one that defends against or inhibits at least 60% of phage infections (e.g., including at least 70%, at least 80%, at least 90%, and at least 95%). A completely resistant strain is one that defends against or inhibits all occurrences of phage infection.

[0025] Characteristic Sequence Elements: As used herein, the term “characteristic sequence elements” refers to gene sequence elements found in a bacteriophage that represent a characteristic portion of the bacteriophage targeted by the CRISPR spacer and distinguish them from host sequences (e.g., sequences found in bacterial cells susceptible to the bacteriophage, and / or sequences found in subjects exposed to the therapeutic bacteria described herein). In some embodiments, the presence of characteristic sequence elements correlates with the presence or level of a particular activity or property of the bacteriophage. In some embodiments, the presence (or absence) of characteristic sequence elements defines a particular bacteriophage strain as a member (or not a member) of a particular family or group of such bacteriophages. Characteristic sequence elements typically consist of at least two monomers (e.g., nucleotides). In some embodiments, the characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., adjacently linked monomers). In some embodiments, the characteristic sequence element refers to the sequence of the bacteriophage's protospacer (e.g., the sequence in the bacteriophage that the CRISPR spacer specifically targets).

[0026] Combination Therapy: As used herein, the term “combination therapy” refers to a situation in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of the first regimen are administered before any dose of the second regimen is administered); and in some embodiments, such agents are administered in overlapping drug regimens. In some embodiments, “administration” of combination therapy may include the administration of one or more agents or therapeutic agents in combination to a subject receiving other agents or therapeutic agents. For clarity, in combination therapy, individual agents do not need to be administered together (or even simultaneously) in a single composition; however, in some embodiments, two or more agents or their active portions may be administered together in a composite composition.

[0027] Comparable: As used herein, “comparable” means two or more sets of substances, entities, situations, conditions, etc., which do not have to be identical to each other, but are similar enough to allow comparison between them, such that a person skilled in the art will understand that a conclusion can be reasonably drawn based on observed differences or similarities. In some embodiments, a comparable set of conditions, situations, individuals, or groups features several substantially identical features and one or a few varying features. A person skilled in the art will understand the degree of identity required for two or more such sets of substances, entities, situations, conditions, etc., to be considered comparable in any given context. For example, a person skilled in the art will understand that sets of situations, individuals, or groups are comparable to each other if they feature a sufficient number and variety of substantially identical features to ensure a reasonable conclusion that differences in results or phenomena obtained under or using different sets of situations, individuals, or groups are caused by or indicate the existence of various differences in those features.

[0028] Symbiosis: As used herein, the term “symbiotic” refers to microorganisms that are nonpathogenic to a host subject and are part of the host subject’s normal microbiome. The term “symbiotic bacteria” refers to bacterial cells or populations of bacterial cells obtained from and adapted to or composed for the microbiome of a mammalian subject. Symbiotic bacteria are adapted to colonize or composed for colonization of a mammalian subject, for example, in internal excretions (e.g., saliva, mucus, urine, or feces), on surfaces (e.g., mucous gastrointestinal tract, mouth / pharynx / nose, urogenital tract, skin, anus / rectum, cheek / mouth, or eye), and not adapted or composed for culture in a laboratory environment.

[0029] Complementary: As used herein, the term “complementary” is used in reference to the hybridization of oligonucleotides related by base-pairing rules. For example, the sequence “CAGT” is complementary to the sequence “GTCA”. Complementarity can be partial or complete. Therefore, any degree of partial complementarity is intended to be included within the scope of the term “complementary,” provided that the partial complementarity allows oligonucleotide hybridization. Partial complementarity is when one or more nucleic acid bases do not match according to base-pairing rules. Total or complete complementarity between nucleic acids is when, under base-pairing rules, each and all nucleic acid bases match with another base.

[0030] CRISPR Spacer: As used herein, a “CRISPR” spacer represents a “clustered, regularly spaced short palindromic repeat” spacer, referring to a nucleotide sequence located between multiple (e.g., two or more) short direct repeats (i.e., CRISPR repeats) in a CRISPR array, such nucleotide sequences corresponding to (e.g., complementary to) characteristic sequence elements of an invading bacteriophage. In some embodiments, a CRISPR spacer is located between two identical CRISPR repeats. In some embodiments, a CRISPR spacer is identified by sequence analysis in a DNA stretch located between two CRISPR repeats.

[0031] Dosage Form: Those skilled in the art will understand that the term “dosage form” may be used to refer to physically distinct units of an active substance for administration to a subject (e.g., a therapeutic agent including a population of therapeutic bacteria, e.g., those described herein). Typically, each such unit contains a predetermined amount of the active substance. In some embodiments, such amount is a unit dose (or all thereof) appropriate for administration according to a drug regimen (i.e., a therapeutic drug regimen) that has been determined to correlate with a desired or beneficial outcome when administered to the relevant population. Those skilled in the art will understand that the total amount of a therapeutic composition or active substance to be administered to a particular subject may be determined by one or more attending physicians and may include administration in multiple dosage forms.

[0032] Dosing regimen: Those skilled in the art will understand that the term “dosing regimen” may be used to refer to a set of (typically two or more) unit doses administered individually to a subject, typically at intervals. In some embodiments, a given active ingredient has a recommended dosing regimen which may comprise one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each dose separated from the others by time. In some embodiments, the individual doses are separated from each other by the same length of time, and in some embodiments, the dosing regimen comprises multiple doses, each dose separated by at least two different time intervals. In some embodiments, all doses in the dosing regimen are of the same unit dose. In some embodiments, different doses in the dosing regimen are of different amounts. In some embodiments, the dosing regimen comprises a first dosing at a first dose, followed by one or more additional dosings at a second dose different from the first dose. In some embodiments, the drug regimen includes a first dose at a first dose, followed by one or more additional doses at a second dose equal to the first dose. In some embodiments, the drug regimen correlates with desired or beneficial outcomes when administered within a relevant population.

[0033] Effective dose: The “effective dose” is an amount sufficient to induce a desired biological response, for example, to treat a condition that the subject may be suffering from. As will be understood by those skilled in the art, the effective dose of a composition or the active substance contained in the composition (e.g., the population of therapeutic bacteria described herein) may vary depending on factors such as the desired biological endpoint, the physical, chemical, and / or biological characteristics of the active substance in the composition (e.g., pharmacokinetics and / or cell viability), the condition being treated, and the age and health status of the subject. Effective doses encompass both therapeutic and preventive treatments. For example, when treating a disease or disorder associated with microbiome dysfunction (e.g., dysbiosis), the effective dose may prevent or reduce at least one symptom associated with the disease or disorder associated with microbiome dysfunction (e.g., dysbiosis). In some embodiments, the effective dose may restore a healthy balance of the microbiome in the microbiome of the subject being treated. In some embodiments, the effective dose may reduce or suppress inflammation in the microbiome of the subject being treated. In some embodiments, the effective dose may reduce or inhibit infection of non-pathogenic commensal bacteria by bacteriophages. Those skilled in the art will understand that the effective dose does not need to be contained in a single dosage form. Rather, the effective dose may, in some cases, require the administration of multiple doses over time (e.g., according to a drug regimen).

[0034] Manipulated: Generally, the term “manipulated” refers to an embodiment that has been manipulated by human hands. For example, in some embodiments, a population of cells or microorganisms (e.g., bacteria) is considered “manipulated” if such population is manipulated to form a desired population of cells or microorganisms, e.g., a concentrated or purified population of therapeutic bacteria as described herein. In some embodiments, a population of cells or microorganisms (e.g., bacteria) is considered “manipulated” if the genetic information of the cells or microorganisms (e.g., bacteria) in the population has been altered (e.g., new genetic material that was not previously present has been introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations, or by mating protocols). As is common practice and as will be understood by those skilled in the art, offspring of cells in a manipulated population are typically still referred to as “manipulated,” even though the actual manipulation has been performed on the previous entities.

[0035] Concentrated: As used herein, the term “concentrated” refers to an increase in the proportion of one or more components of a composition. For example, the therapeutic compositions described herein are concentrated in therapeutic bacteria that are non-pathogenic and symbiotic in the subject to which they are administered and resistant to one or more target bacteriophages. In some such embodiments, the therapeutic compositions described herein contain, for example, at least 10% (e.g., including at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more) higher proportions of therapeutic bacteria (e.g., those described herein) than those of a reference composition (e.g., a fecal sample composition). In some embodiments, the therapeutic compositions described herein contain at least 70% or more (e.g., including at least 80%, at least 90%, at least 95%, or up to 100%) of therapeutic bacteria (e.g., those described herein) relative to all microorganisms present in such therapeutic composition.

[0036] Host: As used herein, the term “host” refers to a subject exposed to a population of therapeutic bacteria (e.g., those described herein) or a therapeutic composition (e.g., those described herein). In some embodiments, the host is a subject suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction. In some embodiments, the host is a subject suffering from or susceptible to an infection by a specific bacteriophage associated with a disease, disorder, or condition. In some embodiments, the host is a subject in which a particular endogenous non-pathogenic symbiotic bacterium is engineered to be resistant to one or more bacteriophages.

[0037] Increase, induce, or decrease: As used herein, these terms or grammatically equivalent comparative terms indicate values ​​that are relative to a comparable reference measurement. For example, in some embodiments, an evaluation value or characteristic achieved in a therapeutic bacterium may be “increased” compared to one obtained in a comparable reference bacterium (e.g., a bacterium not resistant to one or more target bacteriophages). Alternatively or additionally, in some embodiments, an evaluation value or characteristic achieved in a subject may be “increased” compared to one obtained in the same subject under different conditions (e.g., before or after an event; or in the presence or absence of an event such as administration of the therapeutic bacterium population and / or therapeutic composition described herein) or in a different comparable subject (e.g., in a comparable subject different from the subject of interest in terms of prior exposure to the conditions, e.g., without administration of the therapeutic bacterium population and / or therapeutic composition described herein). In some embodiments, comparative terms refer to a statistically relevant difference (e.g., a prevalence and / or magnitude sufficient to achieve a statistical relevance). A person skilled in the art would know, or be able to easily determine, the degree of difference and / or prevalence necessary or sufficient to achieve such statistical significance in a given context.

[0038] Inhibit: In the context of the risk and / or incidence of diseases or bacteriophage infections associated with microbiome dysfunction, the term “inhibit” or “suppress” is not limited to complete inhibition. Therefore, in some embodiments, partial inhibition or relative reduction is included within the scope of the term “inhibit.” In some embodiments, the term refers to a reduction in the risk or incidence of diseases or bacteriophage infections associated with microbiome dysfunction to a level that is reproducible and / or statistically significantly lower than an initial or other appropriate reference level, which may be the baseline level of the risk or incidence of diseases or bacteriophage infections associated with microbiome dysfunction in the absence of or before administration of the compositions described herein. In some embodiments, this term refers to a reduction in the risk or incidence of a microbiome dysfunction-related disease or bacteriophage infection to a level that is, for example, less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level that may be the baseline level of the risk or incidence of a microbiome dysfunction-related disease or bacteriophage infection in the absence of or before administration of the compositions described herein.

[0039] Isolated or Purified: As used herein, the terms “isolated” or “purified” mean (1) a substance and / or entity that has been separated (either in nature or in a laboratory environment) from at least some of the components to which it was originally associated when it was first produced, and / or (2) a substance and / or entity that has been designed, produced, prepared, and / or manufactured by human hands. In some embodiments, the isolated substance or entity may be concentrated, and in some embodiments, the isolated substance or entity may be pure. In some embodiments, the isolated substance and / or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components to which they were originally associated. In some embodiments, the isolated active substance is ultrapure of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may be considered “concentrated,” “isolated,” or even “pure” even after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), in such embodiments, the isolation rate or purity of the substance is calculated without including such carriers or excipients. Those skilled in the art are aware of the various techniques for isolating (e.g., concentrating or purifying) a substance or active substance (e.g., using one or more of fractionation, extraction, precipitation, or other separations).

[0040] Microbiota: As used herein, the term “microbiota” refers to the collective colonization of a body part of interest by a diverse ensemble of microorganisms. Humans have bacterial clusters in different parts of the body, such as the surface or deep layers of the skin (cutaneous microbiota), the mouth (oral microbiota), and the vagina (vaginal microbiota). See Huttenhower C. et al. “Structure, function and diversity of the healthy human microbiome” Nature (2012) 486:207-14 (its entire contents are incorporated herein by reference for the purposes described herein). For example, in some embodiments, microbiota includes the “gut microbiota” or “gut flora,” which refers to the population of microorganisms present in the digestive tract of interest. Such gut microbiota typically contains tens of trillions of microorganisms, including at least 1,000 different known species of bacteria, each with more than 3 million genes (150 times more than human genes). As will be understood by those skilled in the art, certain species of the human gut microbiota are common to a population of human subjects, while certain other species may be individual-specific.

[0041] To modulate: As used in the context of modulating the microbiome, the term “modulate” or “regulate” refers to an entity whose presence or level in a system where a desired activity is observed correlates with a change in the level and / or nature of that activity compared to what would be observed under other equivalent conditions in the absence of such regulation. In some embodiments, regulation refers to increasing the activity and / or level in one or more specific populations of non-pathogenic symbiotic bacteria in the presence of a therapeutic bacterial population and / or therapeutic composition (e.g., as described herein) compared to what would be observed under other equivalent conditions in the absence of the therapeutic bacterial population and / or therapeutic composition (e.g., as described herein). In some embodiments, regulation refers to decreasing the activity and / or level in a target bacteriophage that depletes non-pathogenic symbiotic bacteria in the presence of a therapeutic bacterial population and / or therapeutic composition (e.g., as described herein) compared to what would be observed under other equivalent conditions in the absence of the therapeutic bacterial population and / or therapeutic composition (e.g., as described herein). In some embodiments, regulation refers to a direct interaction with the target entity whose activity is of interest. In some embodiments, regulation refers to an indirect interaction with the target entity whose activity is of interest (i.e., a direct interaction with an intermediate agent that interacts with the target entity). In some embodiments, regulation refers to a change in the level of the target entity of interest, and alternatively or additionally, in some embodiments, regulation refers to a change in the activity of the target entity of interest that does not affect the level of the target entity. In some embodiments, regulation refers to a change in both the level and activity of the target entity of interest, thereby the observed difference in activity is not entirely explained by or equal to the observed difference in level.

[0042] Mutants: As used herein, the term “mutant” refers to an organism, cell, or biomolecule (e.g., nucleic acid or protein) that contains genetic variations compared to a reference organism, cell, or biomolecule. For example, mutant nucleic acids may, in some embodiments, contain mutations, e.g., nucleic acid base substitutions, deletions of one or more nucleic acid bases, insertions of one or more nucleic acid bases, inversions of two or more nucleic acid bases, or cleavages, compared to a reference nucleic acid molecule. Similarly, mutant proteins may contain amino acid substitutions, insertions, inversions, or cleavages, compared to a reference polypeptide. Additional mutations, e.g., fusions and indels, are known to those skilled in the art. Organisms or cells that contain or express mutant nucleic acids or polypeptides may also be referred to herein as “mutants.” In some embodiments, mutants include genetic variants associated with loss of function of a gene product. Loss of function may be complete abolition of function, e.g., abolition of enzymatic activity of an enzyme, or partial loss of function, e.g., decreased enzymatic activity of an enzyme. In some embodiments, mutants include genetic variants associated with improvement of function, e.g., negative or undesirable changes in the characteristics or activity of a gene product. In some embodiments, the mutant is characterized by a decrease or loss of a desired level or activity compared to the reference, and in some embodiments, the mutant is characterized by an increase or boost of an undesirable level or activity compared to the reference. In some embodiments, the reference organism, cell, or biomolecule is a wild-type organism, cell, or biomolecule.

[0043] Non-pathogenic: As used herein, the term “non-pathogenic” refers to a microorganism (e.g., bacteria) that is considered harmless and not associated with a disease, disorder, or condition. In some embodiments, a non-pathogenic microorganism is a beneficial bacterium present in a particular body part of the subject, e.g., the intestines or skin. In some embodiments, a non-pathogenic microorganism may be an opportunistic pathogen in an immunocompromised host, for example. In some embodiments, a non-pathogenic bacterium is not Escherichia coli. In some embodiments, a non-pathogenic bacterium is not Streptococcus.

[0044] Nucleic acid: As used herein, the term “nucleic acid” refers to a polymer of at least three nucleotides. In some embodiments, the nucleic acid includes DNA. In some embodiments, the nucleic acid includes RNA. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid includes both single-stranded and double-stranded portions. In some embodiments, the nucleic acid includes a backbone comprising one or more phosphodiester bonds. In some embodiments, the nucleic acid includes a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may include a backbone comprising one or more phosphorothioate or 5'-N-phosphoamidite bonds and / or one or more peptide bonds, such as “peptide nucleic acid”. In some embodiments, the nucleic acid includes one or more, or all, of the native residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid includes one or more, or all, of the non-native residues. In some embodiments, non-natural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-romouridine, C5-ruoroulidine, C5-iodouridine, C5-propynyluridine, C5-ropinylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the non-natural residues include one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or polypeptide.In some embodiments, the nucleic acid has a nucleotide sequence containing one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., replication in recombinant cells or systems in vivo or in vitro), or chemosynthesis. In some embodiments, the nucleic acid has at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 The residue lengths are 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or longer.

[0045] Operable-associated: As used herein, the term “operable-associated” refers to a parallel relationship in which the described components are such that they function in the manner intended. For example, a “operable-associated” regulatory element to a functional element is associated in such a way that the expression and / or activity of the functional element is achieved under conditions that are compatible with the regulatory element. In some embodiments, the “operable-associated” regulatory element is contiguous (e.g., covalently bonded) with the coding element of interest, and in some embodiments, the regulatory element acts trans to or otherwise from the functional element of interest. In the context of a CRISPR system (e.g., a CRISPR-Cas system), a CRISPR spacer operable-associated to a Cas (CRISPR-associated) polypeptide within a CRISPR locus is associated such that a functional guide RNA containing such a CRISPR spacer is generated to interact with the Cas polypeptide for cleavage and degradation of a target sequence complementary to the CRISPR spacer.

[0046] Pharmaceutical Composition: As used herein, the term “pharmaceutical composition” means a composition in which an active ingredient (e.g., a population of therapeutic bacteria described herein) is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active ingredient (e.g., a population of therapeutic bacteria described herein) is present in a unit dose appropriate for administration in a therapeutic regime that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to the population of interest. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those adapted for: oral administration, e.g., drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g., for oral, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, capsules, etc. In some embodiments, the active ingredient may be therapeutic bacteria (e.g., those described herein) or a population of therapeutic bacteria (e.g., a population of a single therapeutic bacterial species or a mixture of different therapeutic bacterial species), or may contain them. In some embodiments, the active ingredient may be an isolated, purified, or pure population of therapeutic bacteria (e.g., a population of a single therapeutic bacterial species or a mixture of different therapeutic bacterial species), or may contain such populations. In some embodiments, the active ingredient may be a natural product (isolated from a natural source or produced in vitro), or may contain such a product.

[0047] pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable,” which may be used, for example, with respect to a carrier, diluent, or excipient used to formulate a pharmaceutical composition disclosed herein, means that the carrier, diluent, or excipient is compatible with the other components of the composition and is not harmful to its recipient.

[0048] Pharmacopoeia-acceptable carrier: As used herein, the term “pharmacopoeia-acceptable carrier” means a pharmacopoeia-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in transporting or carrying the compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and must not be harmful to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses and their derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; water free of pyrogens; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyacid anhydrides; and other non-toxic and suitable substances used in pharmaceutical formulations.

[0049] Prevention: As used herein, the term “prevention” refers to the delay in the onset of one or more symptoms of a particular disease, disorder, or condition, and / or a reduction in their frequency and / or severity. In some embodiments, the evaluation of prevention is performed on a population basis, such that a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of a particular disease, disorder, or condition is observed in a population susceptible to that disease, disorder, or condition, and the active ingredient is considered to “prevent” that disease, disorder, or condition. In some embodiments, prevention may be considered complete when the onset of the disease, disorder, or condition is delayed over a predetermined period of time.

[0050] Pure: As used herein, a population of cells is “pure” if it is substantially free of other cells and / or components. For example, a preparation containing more than about 90% therapeutic bacteria (e.g., those described herein) is typically considered a pure preparation. In some embodiments, a therapeutic composition is considered “pure” if it contains at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% therapeutic bacteria.

[0051] Reference: As used herein, the term “reference” refers to a standard or control for the subject being compared. For example, in some embodiments, the active substance, animal, individual, population, sample, sequence, or value of interest is compared to the active substance, animal, individual, population, sample, sequence, or value of the reference or control. In some embodiments, the reference or control is tested and / or determined substantially concurrently with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control embodied in a tangible medium, at the discretion of the user. Typically, as will be understood by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances comparable to the subject being evaluated. Those skilled in the art will understand that there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control. In some embodiments, the reference is a negative control reference, and in some embodiments, the reference is a positive control reference.

[0052] Polypeptide: As used herein, the term “polypeptide” typically has the meaning recognized in the art of being a macromolecule of at least three or more amino acids. Those skilled in the art will understand that the term “polypeptide” is intended to be sufficiently general to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides representing functional, biologically active, or characteristic fragments, parts, or domains (e.g., fragments, parts, or domains retaining at least one activity) of such complete polypeptides. In some embodiments, the polypeptide may contain L-amino acids, D-amino acids, or both, and / or any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, the polypeptide may contain native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof (e.g., it may be, or may contain, peptide mimetic substances).

[0053] Prophylactically Effective Dose: A “prophylactically effective dose” is an amount sufficient to prevent a condition (for example, significantly delaying the onset or recurrence of one or more symptoms or characteristics of the condition, so that, for example, it / they are not detected at the time they would be expected without such dose). The prophylactically effective dose of a composition means the amount of therapeutic agent(s) alone or in combination with other agents that provides a preventive effect in preventing a condition. The term “prophylactically effective dose” may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent. Those skilled in the art will understand that a prophylactically effective dose does not need to be contained in a single dosage form. Rather, an effective dose may sometimes require multiple doses administered over time (e.g., according to a dosing regimen).

[0054] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained from or derived from the source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be a cell or organism such as a microorganism, plant, animal, or subject (e.g., human), or may contain them. In some embodiments, the source of interest is a biological sample or contains one. In some embodiments, the biological sample may be amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomit, and / or combinations or components thereof, or may contain them. In some embodiments, the biological fluid may be intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cell permeable fluid, or may contain them. In some embodiments, the biological fluid may be plant exudates, or may contain them. In some embodiments, biological tissue or specimens can be obtained, for example, by aspiration, biopsy (e.g., fine-needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing, or perfusion (e.g., bronchoalveolar, tubal, nasal, ocular, oral, uterine, vaginal, or other washing or perfusion). In some embodiments, the biological specimen is or contains cells obtained from an individual. In some embodiments, the specimen is a “primary specimen” obtained directly from the source of interest by any suitable means. In some embodiments, as will be apparent from the context, the term “specimen” refers to a preparation obtained by processing a primary specimen (e.g., by removing one or more components of the primary specimen and / or by adding one or more active substances to the primary specimen), for example, by filtering using suitable means in the art (e.g., centrifugation and / or semipermeable membrane).Such “processed sample” may include, for example, a specific bacterial or viral fraction isolated from the sample. In some embodiments, the “processed sample” may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques such as nucleic acid amplification or reverse transcription, isolation and / or purification of a specific component.

[0055] Target: The “targets” to which administration is intended include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or non-human animals, e.g., mammals (e.g., primates (e.g., crab-eating macaques, rhesus macaques); domestic animals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds (e.g., chickens, ducks, geese, and / or turkeys)). In certain embodiments, the animal is a mammal (e.g., at any developmental stage). In some embodiments, the animal (e.g., a non-human animal) may be a transgenic or genetically modified animal. In some embodiments, the target is a mammalian subject (e.g., a human subject). In some embodiments, the target is a mammalian subject suffering from a disease, disorder, or condition associated with dysbiosis.

[0056] Affected: An individual “affected” with a disease, disorder, and / or condition is diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0057] Susceptible: An individual who is “susceptible” to a disease, disorder, and / or condition is an individual who is at a higher risk of developing that disease, disorder, and / or condition than a member of the general population. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not be diagnosed with that disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of that disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of that disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may develop that disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not develop that disease, disorder, and / or condition.

[0058] Symptoms are reduced: As used herein, "symptoms are reduced" means that one or more symptoms of a particular disease, disorder, or condition are reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency. For the purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

[0059] Therapeutic bacteria: As used herein, the term “therapeutic bacteria” refers to bacteria that, when administered to a subject, have a therapeutic effect and / or induce a desired biological and / or pharmacological effect. In some embodiments, therapeutic bacteria or populations of therapeutic bacteria include non-pathogenic symbiotic bacteria that can be used to alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition associated with dysbiosis or microbiome dysfunction.

[0060] Therapeutic Dose: A “therapeutic dose” is a quantity sufficient to provide a therapeutic effect in the treatment of a condition, which may include, for example, a reduction in frequency and / or severity and / or a delay in the onset of one or more characteristics or symptoms associated with the condition. A therapeutic dose means the amount of a therapeutic agent (e.g., therapeutic bacteria) alone or in combination with other therapies that provides a therapeutic effect in the treatment of a condition. A “therapeutic dose” may include an amount that improves the overall treatment, reduces or avoids the symptoms or causes of the condition, or enhances the therapeutic effect of another therapeutic agent. Those skilled in the art will understand that a therapeutic dose does not necessarily have to be contained in a single dosage form. Rather, an effective dose may, in some cases, require the administration of multiple doses over time (e.g., according to a dosing regimen).

[0061] Treatment: The terms “treatment,” “to treat,” and “to treat” mean to reduce, alleviate, delay the onset of, or inhibit the progression of a “pathological condition” (e.g., a disease, disorder, or state including one or more signs or symptoms thereof) as described herein, such as a disease, disorder, or state associated with dysbiosis or microbiome dysfunction (e.g., inflammatory bowel disease). In some embodiments, treatment may be administered after one or more signs or symptoms have developed or been observed. Treatment may be continued after the symptoms have subsided, for example, to delay or prevent recurrence and / or spread.

[0062] Variant: As used herein, the term “variant” refers to an entity that exhibits significant structural identity with a reference entity but is structurally different from the reference entity in the presence or level of one or more chemical parts. In many embodiments, a variant is also functionally different from its reference entity. Generally, whether a particular entity is considered a “variant” of a reference entity depends on the degree of structural identity with that reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a variant is a distinct chemical entity that shares one or more such characteristic structural elements. To give only a few examples, small molecules may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant portions, so that variants of small molecules share the core structural element and characteristic pendant portions but differ in the other pendant portions and / or the types of bonds present in the core (e.g., single-paired double, E-paired Z), polypeptides may have characteristic sequence elements consisting of multiple amino acids having designated positions in a linear or three-dimensional space and / or contributing to a specific biological function, and nucleic acids may have characteristic sequence elements consisting of multiple nucleotide residues having designated positions in a linear or three-dimensional space. For example, a variant therapeutic bacterium may differ from a reference therapeutic bacterium as a result of one or more structural modifications (e.g., addition, deletion, and / or modification of chemical moieties), provided that the variant therapeutic bacterium is resistant to bacteriophages that target bacteria associated with a disease, disorder, or condition.In some embodiments, the variant therapeutic bacteria are characterized in that, when evaluated in vitro by culturing a population of such variant therapeutic bacteria in the presence of one or more target bacteriophages after 24 hours or more (e.g., including 48 hours, 72 hours, or more), the cellular viability of such variant therapeutic bacteria is at least 60% (e.g., including at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or up to 100%) of that observed when a reference therapeutic bacteria is cultured in the presence of one or more target bacteriophages. In some embodiments, the variant therapeutic bacteria are characterized in that, when evaluated in vitro by culturing a population of such variant therapeutic bacteria in the presence of one or more target bacteriophages after 24 hours or more (e.g., including 48 hours, 72 hours, or more), the cellular viability of such variant therapeutic bacteria is at least 1.1 times (e.g., including at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 2 times, or more) than that observed when a reference therapeutic bacterium is cultured in the presence of one or more target bacteriophages. In some embodiments, the variant therapeutic bacteria exhibit at least one physical characteristic that differs from the reference therapeutic bacterium. For example, in some embodiments, the variant therapeutic bacteria may have genetic alterations in biological pathways compared to the reference therapeutic bacterium. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 structural modification compared to the reference. In some embodiments, the variant has a small number of structural modifications (e.g., five, four, three, two, or fewer than one). In some embodiments, the variant has additions or deletions of no more than five, four, three, two, or one chemical moieties compared to the reference, and in some embodiments, it has no additions or deletions. In some embodiments, the variant is an entity that can be produced from the reference by a chemical operation.In some embodiments, the variant is an entity that can be produced by performing a synthesis process that is substantially similar to the one that produces the reference (for example, sharing multiple steps).

[0063] Vectors: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is bound. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which additional DNA segments may be ligated. Another type of vector is a viral vector, to which additional DNA segments may be ligated into a viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can lead to the expression of a gene to which they are functionally linked.

[0064] Standard techniques may be used for recombinant DNA, nucleic acid synthesis, e.g., DNA template synthesis and / or RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures may generally be carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory See Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (this is incorporated herein by reference for all purposes)).

[0065] This disclosure provides, in particular, technologies relating to therapeutic compositions comprising or containing phage-resistant bacteria, as well as various related methods and / or materials, including, for example, methods of administering such compositions to treat diseases or disorders associated with the microbiome.

[0066] In some embodiments, the technology provided is more useful than certain existing technologies, such as the administration of conventional probiotics and / or beneficial bacteria, in treating microbiome-related diseases or disorders. For example, this disclosure understands that many such conventional probiotic (bacteria)-based therapies may be less effective, or even ineffective, in treating or preventing diseases or disorders associated with dysbiosis or microbiome dysfunction. Specifically, this disclosure recognizes, among other things, that bacteriophages present in the human gut can infect and deplete the healthy and beneficial bacterial populations present in the gut of humans suffering from inflammatory bowel disease (IBD). Thus, this disclosure provides insight, among other things, into the existence of predatory interactions between bacteriophages and bacteria in certain human body sites, and therefore, the presence of bacteriophages that infect and deplete beneficial symbiotic bacteria may significantly impair the effectiveness of conventional probiotic (bacteria)-based therapies. This disclosure provides, in particular, techniques for solving such problems, including therapeutic bacteria, compositions, and methods, which include, for example, specifically exposing a target in need to a population of non-pathogenic symbiotic bacteria that is resistant to one or more target bacteriophages associated with a disease or disorder related to dysbiosis or microbiome dysfunction.

[0067] I. Therapeutic bacteria This disclosure provides therapeutic bacteria that (i) are non-pathogenic and symbiotic in the target being treated, and (ii) are resistant to one or more bacteriophages that target (e.g., selectively target) the corresponding non-pathogenic and symbiotic host bacteria in the target. Those skilled in the art will recognize, upon reading this disclosure, that therapeutic bacteria are useful for treating and / or preventing diseases or disorders associated with dysbiosis or the microbiome.

[0068] A. Nonpathogenic and symbiotic bacteria The microbiome may include various non-pathogenic and symbiotic bacterial species, any one of which may be used in accordance with this disclosure. In some embodiments, the genus and / or species of non-pathogenic symbiotic bacterial cells may depend on the specificity of the bacteriophage (e.g., phage host range). For example, some bacteriophages exhibit tropism or preferential targeting of certain bacterial species.

[0069] Bacteria are typically small (typical linear dimensions are around 1 micron), uncompartmentalized, and contain circular DNA and 70S ribosomes. In some embodiments, nonpathogenic and symbiotic bacteria include bacteria from the subclassifications of Eubacteria and Archaeobacteria. Eubacteria can be further subdivided into Gram-positive Eubacteria and Gram-negative Eubacteria, depending on differences in cell wall structure. Those classified based solely on macroscopic morphology (e.g., cocci, bacilli) are also included herein. In some embodiments, nonpathogenic and symbiotic bacteria are Gram-negative cells or include them. In some embodiments, nonpathogenic and symbiotic bacteria are Gram-positive cells or include them.Non-limiting examples of non-pathogenic and commensal bacteria useful in accordance with the present disclosure include Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, such as Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus Examples of bacteria include those in the groups *paracasei*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Lactococcus lactis* (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976).

[0070] In some embodiments, the non-pathogenic and symbiotic bacteria used in the therapeutic bacteria may be one or more (e.g., two or more, three or more, four or more, five or more) of the following bacteria, or may include them: Clostridium symbiosum, Clostridium hathewayi, Clostridium citroniae, Clostridium bolteae, Ruminococcus species M-1, Ruminococcus gnavus, Blautia species Canine oral taxon 143, Anaerostipes caccae, Clostridium lactatifermentans, Coprobacillus cateniformis, Clostridium ramosum, cf. Clostridium species MLG055, Clostridium innocuum, Eubacterium desmolans, Clostridium orbiscindens, Ruminococcus species 16442, Anaerotruncus colihominis, Bacteroides dorei, Bifidobacterium pseudolongum subspecies Pseudolongum, Bifidobacterium breve, Clostridium clostridioforme, Clostridium collagenovorans, Clostridium asparagiforme, Clostridium scindens, Clostridium acetireducens, Clostridium algidicarnis, Clostridium paradoxum, Clostridium saccharogumia, Clostridium ramosum JCM1298, Clostridia UC5.1-1A9, Clostridium asparagiforme, Clostridium cellulosi, Clostridium bolteae, Clostridium citroniae, Clostridium clostridioforme, Clostridium indolis, Clostridium cocleatum, Clostridium innocuum, Clostridium lavalense, Clostridium saccharolyticum, Clostridium scindens, Clostridium symbiosum, Clostridium butyricum, Clostridia bacterium UC5.1-1A9, Clostridium jeddahense, Clostridium nigeriense, Clostridium neonatale, Clostridium perfringens, Clostridium phoceensis, Clostridium species 1_1_41A1FAA, Clostridium species 316002 / 08, Clostridium species 7_3_54FAA, Clostridium species ATCC BAA-442, Clostridium species C105KSO14, Clostridium species CL-6, Clostridium species D5, Clostridium species FS41, Clostridium species HGF2, Clostridium species IODB-O3, Clostridium species KLE 1755, Clostridium species L2-50, Clostridium species M62 / 1, Clostridium species MSTE9, Clostridium species VE202-10, Clostridia bacterium UC5.1-2G4, Clostridia bacterium UC5.1-2H11, Clostridium species 1_7_47FAA, Clostridium species JGI 000176CP_D02, Clostridium species VE202-03, Clostridium species VE202-06, Clostridium species VE202-07, Clostridium species VE202-09, Clostridium species VE202-15, Clostridium species VE202-16, Clostridium species VE202-21, Clostridium species VE202-26, Clostridium species VE202-27, Clostridium species VE202-28, Clostridium species VE202-29, Clostridium species C8, Clostridium. sporogenes, Clostridium tyrobutyricum, Clostridium species VE202-10, Lachnoclostridium species An131, Lachnoclostridium species YL32, Lachnospiraceae 3_1_57FAA_CT1, Lachnospiraceae 5_1_57FAA, Lachnospiraceae 6_1_63FAA, Lachnospiraceae A4, Lachnospiraceae DJF_VP30, Lachnospiraceae VE202-23, Lachnospiraceae VE202-23, Clostridium acetireducens, Clostridium collagenovorans, and combinations thereof.

[0071] In some embodiments, the non-pathogenic and symbiotic bacteria are one or more species of Clostridia, or include them. Exemplary species of Clostridia include, but are not limited to, Clostridium scindens, Clostridiales, Clostridium symbosium, Clostridiales bacterium, Clostridium phoceensis, Clostridium innocuum, and combinations thereof. In some embodiments, the non-pathogenic and symbiotic bacteria are Clostridium scindens, or include it.

[0072] In some embodiments, the nonpathogenic and symbiotic bacteria are one or more Bifidobacteria species. In some embodiments, the nonpathogenic and symbiotic bacteria are Bifidobacterium longum, or include it.

[0073] In some embodiments, non-pathogenic and symbiotic bacteria include Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus This includes, but is not limited to, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus buchneri, and Lactobacillus sakei, but is one or more species of Lactobacillus, or includes them.

[0074] In some embodiments, the nonpathogenic and symbiotic bacteria are one or more species of Akkermansia. In some embodiments, the nonpathogenic and symbiotic bacteria are Akkermansia muciniphila or include it.

[0075] In some embodiments, the therapeutic bacteria include one or more non-pathogenic and symbiotic bacteria (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) that are found to be normally present in a body site of a human subject (e.g., mucosal gastrointestinal tract, mouth / pharynx / nasal cavity, urogenital tract, skin, anus / rectum, cheek / mouth, or eye). In some embodiments, the therapeutic bacteria include one or more variants (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of non-pathogenic and symbiotic bacteria that are found to be normally present in a body site of a human subject (e.g., mucosal gastrointestinal tract, mouth / pharynx / nasal cavity, urogenital tract, skin, anus / rectum, cheek / mouth, or eye).

[0076] In some embodiments, the therapeutic bacteria population includes at least one isolated, purified, or cultured symbiotic bacteria (e.g., including at least two, at least three, at least four, at least five, or more) selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Coprococcus, Clostridium, Collinsella, Desulfomonas, Dorea, Escherichia, Eubacterium, Fusobacterium, Gemmiger, Lactobacillus, Lactoccucs, Monilia, Peptostreptococcus, Propionibacterium, Ruminococcus, and combinations thereof. In some embodiments, the therapeutic bacteria population includes Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, Lactoccucs, or combinations thereof. In some embodiments, such bacteria may be of self-derived origin (for example, the therapeutic bacteria described herein are genetically engineered from a target bacteriophage susceptible to infection by the target bacteriophage). In some embodiments, such bacteria may be homogeneous.

[0077] B. bacteriophage In particular, the present disclosure provides therapeutic compositions for non-pathogenic and symbiotic bacteria that are established to be resistant to one or more bacteriophages that would otherwise infect such non-resistant non-pathogenic and symbiotic bacteria.

[0078] Bacteriophages (also known as phages) are typically composed of proteins that encapsulate DNA or RNA genomes, which may encode only a few or a few hundred genes, thereby producing virions with relatively simple or detailed structures. Therefore, bacteriophages are one of the most common and diverse entities in the biosphere. Phages are classified according to the International Committee on Taxonomy of Viruses (ICTV), taking into account their morphology and nucleic acid type (DNA or RNA, single-stranded or double-stranded, linear or circular). Approximately 19 phage families that infect bacteria and / or archaea (the prokaryotic domain previously classified as archaebacteria) have been recognized to date. Many bacteriophages are specific to bacterial cells of a particular genus, species, or strain.

[0079] In some embodiments, non-pathogenic and symbiotic bacteria used in the compositions and / or methods described herein may be resistant to lytic bacteriophages. A lytic bacteriophage follows the lytic pathway through the completion of a lytic cycle, rather than entering the lysogenic pathway. The lytic bacteriophage undergoes viral replication, which leads to the lysis of the cell membrane, cell destruction, and the release of progeny bacteriophage particles that can infect other cells.

[0080] In some embodiments, non-pathogenic and symbiotic bacteria used in the compositions and / or methods described herein may be resistant to lysogenic bacteriophages. Lysogenic bacteriophages are those that can enter the lysogenic pathway, becoming dormant passive portions of the cell genome before their lysis cycle is complete.

[0081] In some embodiments, non-pathogenic and symbiotic bacteria used in the compositions and / or methods described herein may be resistant to the Temperate bacteriophage. Temperate is a phage that can be soluble or lysogenic. In the lysogenic case, such a phage typically maintains a quiescent state, integrating its nucleic acid into the host cell genome and replicating only when the host genome is replicated. In its soluble or plant-growing phage, the phage nucleic acid either cleaves itself from the host genome or is not integrated into the host cell genome, but rather takes over the host cell's protein synthesis mechanism at the expense of cellular components to assemble the phage progeny. The new phage is released from the infected host cell when the cell is lysed.

[0082] In some embodiments, the non-pathogenic and symbiotic bacteria used in the compositions and / or methods described herein are resistant to bacteriophages, which are toxic to the bacterial cells at some point in their life cycle after infection.

[0083] While non-pathogenic and symbiotic bacteria may be used to confer resistance to any target bacteriophage (including, for example, wild-type, naturally occurring, isolated, or recombinant bacteriophages) in accordance with this disclosure, in some embodiments, target bacteriophages that are active (e.g., capable of infecting) one or more non-pathogenic symbiotic bacterial strains in the microbiome of a mammalian subject (e.g., human) are of particular interest. As merely an example, in some embodiments, target bacteriophages to which non-pathogenic and symbiotic bacteria are resistant include, but are not limited to, bacteriophages capable of infecting bacteria from at least one of the following genera: Bacillus, Bacteroides, Bifidobacterium, Clostridium, Collinsella, Coprococcus, Desulfomonas, Dorea, Escherichia (e.g., E. coli), Eubacterium, Fusobacterium, Gemmiger, Monilia, Lactobacillus, Peptostreptococcus, Propionibacterium, Akkermansia, and Ruminococcus.

[0084] In some embodiments, the therapeutic bacteria described herein are resistant to one or more bacteriophages selected from the group consisting of Caudovirales or Microviridae phages.

[0085] In some embodiments, the therapeutic bacteria described herein are resistant to one or more bacteriophages present in the gut microbiome of human subjects. For example, in some such embodiments, the therapeutic agents described herein are resistant to one or more bacteriophages, which may be Caudovirales or contain them.

[0086] C. Exemplary bacteriophage-resistant bacteria The therapeutic bacteria used in accordance with this disclosure are resistant to one or more bacteriophages (e.g., those described herein). In some embodiments, the therapeutic bacteria are resistant to one or more bacteriophages that would otherwise infect the corresponding bacteria without such phage resistance. In some such embodiments, the bacteriophages against which the therapeutic bacteria are resistant are associated with diseases, disorders, or conditions related to dysbiosis and / or microbiome dysfunction.

[0087] In some embodiments, bacteriophage-resistant bacteria can be isolated from biological tissue or fluid samples of a subject (e.g., a mammalian subject). For example, in some embodiments, bacteriophage-resistant bacteria may be isolated from internal excretions of a mammalian subject, including, but not limited to, saliva, mucus, urine, and / or feces (e.g., fecal samples). In some embodiments, bacteriophage-resistant bacteria can be genetically engineered in vitro or ex vivo. In some embodiments, bacteriophage-resistant bacteria can be generated in vivo, for example, by administering a composition comprising a nucleic acid sequence to a subject, the nucleic acid sequence being delivered to a host bacteriophage susceptible to such bacteriophage for genetic engineering to make it resistant to such bacteriophage.

[0088] 1. CRISPR systems (e.g., CRISPR-Cas systems) In some embodiments, therapeutic bacteria for use in the compositions and / or methods described herein each comprise a clustered, regularly spaced, short-palindrical repeat (CRISPR) system comprising one or more bacteriophage sequences and CRISPR spacers targeting CRISPR-associated (Cas) polypeptides. In some such embodiments, the CRISPR system is characterized in that, when the therapeutic bacteria are infected with a bacteriophage comprising a target spacer sequence (protospacer) or an associated variant of such target spacer sequence (e.g., in some embodiments, a mutant bacteriophage having a protospacer that evolves at least one, e.g., at least two, at least three, or at least four mutations from a parent or target bacteriophage; or in some embodiments, a mutant bacteriophage having a protospacer that evolves one to four mutations from a parent or target bacteriophage), the therapeutic bacteria exhibit increased resistance to infection compared to that observed for otherwise equivalent bacteria that do not contain CRISPR spacers targeting the bacteriophage or its associated variant. In some embodiments, such therapeutic bacteria can exhibit at least 30% or more (e.g., including at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more) increased resistance to bacteriophage infection compared to those observed for otherwise equivalent bacteria that do not contain a CRISPR spacer targeting a bacteriophage or an associated variant. In some such embodiments, bacteriophage resistance may be characterized by one or more methods described in the following section entitled “Exemplary Methods for Characterizing Bacteriophage Resistance.”

[0089] The CRISPR-Cas system has evolved within bacteria to provide adaptive immunity against foreign genetic elements, including phages. CRISPRs are typically short, partially palindromic sequences of 24–65 bp containing inner and terminal inversion repeats. While isolated elements have been detected, they are generally arranged in clusters of repeat units (up to approximately 20 or more per genome) spaced apart by unique intervening 20–58 bp sequences.

[0090] CRISPR systems are found in approximately 40% and 90% of sequenced bacterial and archaeal genomes, respectively. Diverse arrays of CRISPR systems have also been identified in non-pathogenic and symbiotic bacteria. (Rho, M., et al. PLoS Genet. 8, e1002441 (2012), Soto-Perez, P. et al. Cell Host Microbe 26, 325-335. e5 (2019)). CRISPR systems present in non-pathogenic and symbiotic bacteria that can be operated according to this disclosure include, for example, type IA, type IB, type IC, type ID, type IE, type IF, type III-A, type III-B, type II-A, or type II-B. In some embodiments, the CRISPR systems present in non-pathogenic and symbiotic bacteria that can be operated according to this disclosure are type I (e.g., type IC) or type III (e.g., type III-A, type III-B). Various computer software and web resources are available for the analysis and identification of CRISPR systems and CRISPR arrays that may be useful in the compositions and / or methods described herein. These tools include, but are not limited to, CRISPR detection software such as PILERCR, CRISPR recognition tools, and CRISPRFinder; online repositories of pre-analyzed CRISPRs such as CRISPRdb; and tools for browsing CRISPRs in microbial genomes such as Pygram. As will be understood by those skilled in the art, a CRISPR array from any one of such CRISPR systems may be used in accordance with this disclosure. In some embodiments, the therapeutic bacteria described herein may use an endogenous CRISPR array to modulate cellular resistance to a target bacteriophage. In some embodiments, the therapeutic bacteria described herein may include a heterologous CRISPR array comprising at least one Cas protein (e.g., one described herein), at least one CRISPR spacer (e.g., one described herein), and at least two CRISPR repeats introduced therein (e.g., one described herein) to modulate cellular resistance to a target bacteriophage.

[0091] CRISPR Spacers: Each therapeutic bacterium used in accordance with this disclosure comprises one or more CRISPR spacers targeting one or more bacteriophage sequences, e.g., one or more characteristic sequence elements of a bacteriophage(s). In some embodiments, such CRISPR spacers may target characteristic nucleic acid sequence elements or transcripts of one or more bacteriophages. Such CRISPR spacers may be naturally present in bacterial cells, or may be endogenously expressed, or may be introduced into such cells by methods known in the art.

[0092] In some embodiments, the therapeutic bacteria described herein include, for example, at least one bacterium comprising at least two, at least three, at least four, at least five, or more CRISPR spacers, each of which targets a different characteristic sequence element (e.g., a characteristic nucleic acid sequence element or its transcript) of the same target bacteriophage.

[0093] A CRISPR spacer is typically a sequence that is complementary to a characteristic sequence element of a target bacteriophage (e.g., a characteristic nucleic acid sequence element or its transcript, or a protospacer), and is therefore capable of binding to a characteristic sequence element of one or more bacteriophages or their associated variants, resulting in cleavage of such characteristic sequence element in the presence of a suitable Cas polypeptide, or includes such a sequence. In some embodiments, the CRISPR spacer is a sequence that is complementary (e.g., 100% complementary) to a characteristic sequence element of a reference bacteriophage (e.g., a characteristic nucleic acid sequence element or its transcript, or a protospacer), or includes such a sequence. In some embodiments, the CRISPR spacer is a sequence having at least one base pair mismatch (e.g., including at least two base pair mismatches, at least three base pair mismatches, at least four base pair mismatches, or more) to a characteristic sequence element of a reference bacteriophage (e.g., a characteristic nucleic acid sequence element or its transcript, or a protospacer), or includes such a sequence. In some embodiments, the CRISPR spacer is a sequence having one to four base pair mismatches with respect to a characteristic sequence element of a reference bacteriophage (e.g., a characteristic nucleic acid sequence element or its transcript, or a protospacer), or includes such a sequence.

[0094] In some embodiments, the CRISPR spacer is a sequence present as a CRISPR spacer within the CRISPR locus of non-pathogenic and symbiotic bacteria found in the human gut, or comprises such a sequence, which typically matches characteristic sequence elements of bacteriophages found in the human gut. For example, in some embodiments, the CRISPR spacer is, for example, Soto-Perez The sequence present as a CRISPR spacer within the CRISPR locus of Eggerthella lenta, a human intestinal Actinobacterium, as described in et al. Cell Host & Microbes (2019) 26:1-11 (the contents of which are incorporated herein by reference in their entirety).

[0095] In some embodiments, the CRISPR spacer is or includes a sequence determined by a computational approach. For example, as described in Example 2, the CRISPR spacer in some embodiments may be determined by matching a portion of a bacteriophage sequence to a known CRISPR spacer sequence identified from a bacterial host, such as Enterobacteriaceae.

[0096] Cas polypeptides or genes encoding them: CRISPR structures or arrays are typically found near CRISPR-related (Cas) genes. Various Cas genes or polypeptides known in the art may be used in the compositions and / or methods described herein, and the selection of Cas polypeptides may vary depending on the bacteriophage targeted by the compositions and / or methods described herein. In some embodiments, Cas proteins may be selected based on their effectiveness in conferring resistance to a bacteriophage population. Examples of Cas polypeptides that may be useful in the compositions and / or methods described herein include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, and combinations thereof. In some embodiments, the therapeutic bacterium may contain a type III Cas polypeptide, e.g., Cas10 polypeptide. In some embodiments, the therapeutic bacterium contains Cas It may contain an RNA nuclease. In some embodiments, the therapeutic bacteria described herein may contain endogenous Cas polypeptides. In some embodiments, the therapeutic bacteria described herein may contain heterologous Cas polypeptides.

[0097] In some embodiments, the therapeutic bacteria described herein may include one or more Cas genes or polypeptides that are endogenous to such therapeutic bacteria, and one or more heterologous CRISPR spacers operably associated with such one or more Cas genes or polypeptides.

[0098] In some embodiments, the therapeutic bacteria described herein may include one or more Cas genes or polypeptides heterogeneous to such therapeutic bacteria, and one or more CRISPR spacers that may be homologous or heterogeneous to such therapeutic bacteria. In some such embodiments, one or more CRISPR spacers may be operably associated with such one or more Cas genes or polypeptides.

[0099] Methods for modulating CRISPR-mediated immunity in cells are known in the art, as described, for example, in U.S. Patent No. 9,879,269 and U.S. 2016 / 0348120, the contents of which are incorporated herein by reference in their entirety for the purposes described herein. Those skilled in the art will recognize, upon reading this disclosure, that such methods and other methods known in the art may be used to generate therapeutic bacteria according to some embodiments described herein.

[0100] 2. Bacteriophage receptor variants In some embodiments, therapeutic bacteria for use in the compositions and / or methods described herein each comprise at least one variant of a bacteriophage receptor(s) on the bacterial cell surface. In some embodiments, one or more mutations in the bacteriophage receptor conferring resistance can be identified by exposing a bacterial population to phages and selecting bacteria that exhibit improved survival. This strategy allows for the isolation of several phage-resistant bacteria having mutations in the bacteriophage receptor(s) on the bacterial cell surface. These mutations can then be manipulated into a desired bacterial strain to confer resistance. In some such embodiments, therapeutic bacteria are genetically engineered to express at least one variant of a bacteriophage receptor(s) on the bacterial cell surface. In some such embodiments, therapeutic bacteria are isolated or purified from a biological sample from a subject. In some embodiments, such therapeutic bacteria may be isolated or purified from fecal samples from individuals determined to be less susceptible to inflammatory bowel disease (e.g., healthy individuals).

[0101] D. Manipulated bacterial populations This disclosure provides, in particular, engineered populations of therapeutic bacteria (e.g., those described herein) that are resistant to one or more bacteriophages. Such populations of therapeutic bacteria may be useful in treating diseases or disorders associated with microbiome dysfunction.

[0102] In some embodiments, the manipulated population of therapeutic bacteria is or comprises a concentrated or purified population of therapeutic bacteria (e.g., those described herein). For example, in some embodiments, naturally occurring phage-resistant bacterial cells are concentrated or purified from a mixed population of bacteria. In some embodiments, such naturally occurring phage-resistant bacterial cells may contain CRISPR spacers that target one or more bacteriophages. In some embodiments, such naturally occurring phage-resistant cells may also have mutations that affect cell wall properties that inhibit phage infection. In some embodiments, such naturally occurring phage-resistant bacterial cells may be isolated or purified from a biological sample of a human subject or a population of human subjects. In some embodiments, such isolated or purified phage-resistant bacterial cells may be cultured in vitro for clonal selection and / or cell proliferation.

[0103] In some embodiments, phage-resistant bacterial cells can be isolated by exposing a susceptible bacterial culture to a target phage. Typically, the majority of bacterial cells (>90%) can be eliminated in the presence of the target phage (e.g., after 24 hours or more, including 48 hours, 72 hours, or longer). Surviving bacterial cells can be considered phage-resistant candidates. In some embodiments, such surviving bacterial cells (after the initial exposure to the target phage) may be subjected to at least a second phage exposure, e.g., exposure to the same target or a different phage, and bacterial cells surviving after such a second phage exposure can be characterized as phage-resistant. Such phage-resistant cells can be cultured in vitro for clonal selection and / or cell proliferation.

[0104] In some embodiments, isolated phage-resistant bacterial cells can be sequenced to identify the mutation(s) or CRISPR spacer that confers resistance. Such information may be useful in some embodiments for genetically engineered phage-resistant bacteria. Thus, in some embodiments, the engineered population of therapeutic bacteria is or includes a population of therapeutic bacteria (e.g., as described herein) that includes non-pathogenic and symbiotic bacteria genetically engineered to be resistant to one or more target bacteriophages, as described in the section above entitled “Exemplary Bacteriophage-Resistant Bacteria”.

[0105] In some embodiments, all therapeutic bacteria in a population (e.g., those described herein) originate from a single strain. In some embodiments, all therapeutic bacteria in a population (e.g., those described herein) originate from a single clone. In some embodiments, the therapeutic bacteria in a population (e.g., those described herein) comprises a collection of bacterial strains. In some embodiments, the therapeutic bacteria in a population (e.g., those described herein) comprises at least one strain found in the human microbiome. In some embodiments, the therapeutic bacteria in a population (e.g., those described herein) comprises genetically modified variants. In some embodiments, the therapeutic bacteria in a population (e.g., those described herein) comprises multiple bacterial strains, each present in the human microbiome, and in some such embodiments, such population comprises individual strains in relative amounts (e.g., different from those found in human populations (the average, and / or specific subpopulations, and / or specific humans or their aggregates) (e.g., different from each other and / or a reference strain).

[0106] In some embodiments, the therapeutic bacteria in a population (e.g., those described herein) are of the same bacterial species, and at least two subsets (e.g., at least three, at least four, at least five, or more) of such therapeutic bacteria contain one or more different gene modifications (e.g., distinct CRISPR spacers and / or distinct bacteriophage receptor variants) that confer resistance to one or more bacteriophages.

[0107] In some embodiments, the therapeutic bacteria in a population (e.g., those described herein) are different bacterial genera and / or species, and each subset of a distinct bacterial genus and / or species comprises one or more distinct gene modifications (e.g., distinct CRISPR spacers and / or distinct bacteriophage receptor variants) that confer resistance to one or more bacteriophages.

[0108] Exemplary methods for characterizing E. bacteriophage resistance Bacterial cells (e.g., isolated from biological samples or genetically engineered) can be evaluated or characterized for bacteriophage resistance in order to identify and select phage-resistant bacteria.

[0109] In some embodiments, a bacterial population (e.g., a clonal population) that is presumed or predicted to have developed resistance to a bacteriophage as a result of isolation and / or manipulation may be cultured in a suitable liquid medium and challenged with a single characterized / uncharacterized bacteriophage, a cocktail of multiple characterized / uncharacterized bacteriophages, or a biological composition likely to contain characterized / uncharacterized bacteriophages that have been shown to infect clonal bacterial populations prior to experimental intervention. Bacteriophage-challenged bacterial cultures that exhibit statistically similar growth to unchallenged bacterial cultures can be considered to have developed complete resistance to the challenged bacteriophage population. Bacteriophage-resistant cultures challenged with a bacteriophage may also have statistically higher growth rates than bacteriophage-susceptible cultures, and these would be characterized as partial resistance. This method can be adapted when clonal and non-clonal bacterial populations can be challenged with a bacteriophage before culturing or during the delay period.

[0110] Additionally or alternatively, bacterial bacteriophage resistance phenotypes and / or genotypes can be identified using solid media via plaque assays. For example, bacteria suspected or predicted to have developed resistance to bacteriophages as a result of experimental intervention (e.g., isolation and / or manipulation) are grown on solid media and challenged with serial dilutions of appropriate buffers containing a single characterized / uncharacterized bacteriophage, a cocktail of multiple characterized / uncharacterized bacteriophages, or a characterized / uncharacterized bacteriophage shown to infect clonal bacterial populations prior to the experimental intervention. Bacterial colonies surviving after bacteriophage challenge can be considered bacteriophage resistant if controls containing the bacteria universally develop plaques prior to the experimental intervention.

[0111] In some embodiments, the underlying genetic mechanisms of bacterial resistance to bacteriophage populations can be determined using comparative genomics. For example, in some embodiments, the genome of a bacterial isolate exhibiting bacteriophage resistance is compared to a bacteriophage-susceptible population of the same bacterial strain.

[0112] II. Exemplary Compositions This disclosure also provides, in particular, compositions that exhibit bacterial resistance to bacteriophages, for example, bacteriophages associated with diseases or disorders related to microbiome dysfunction or dysbiosis. In some embodiments, such compositions may be more useful in treating microbiome-related diseases or disorders than certain existing techniques, for example, the administration of conventional probiotics and / or beneficial bacteria, which can still be infected and depleted by bacteriophages present in the microbiome being treated.

[0113] A. Pharmaceutical compositions containing therapeutic bacteria One embodiment described herein relates to a pharmaceutical or therapeutic composition comprising an engineered population of therapeutic bacteria that (i) is nonpathogenic and symbiotic in the subject to which it is administered, and (ii) is resistant to one or more target bacteriophages. In some embodiments, the pharmaceutical or therapeutic composition comprises an engineered bacterial population as described in the section titled “Engineered Bacterial Population” above.

[0114] In some embodiments, the therapeutic bacteria contained in the pharmaceutical or therapeutic compositions described herein include at least one isolated, purified, or cultured bacteria (e.g., including at least two, at least three, at least four, at least five, or more) selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Coprococcus, Clostridium, Collinsella, Desulfomonas, Dorea, Escherichia, Eubacterium, Fusobacterium, Gemmiger, Lactobacillus, Lactoccucs, Monilia, Peptostreptococcus, Propionibacterium, Ruminococcus, and combinations thereof. In some embodiments, the therapeutic bacteria contained in the therapeutic compositions described herein include Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, Lactoccucs, or combinations thereof. In some embodiments, such bacteria may be autologous. In some embodiments, such bacteria may be homogeneous.

[0115] A pharmaceutically active composition containing nucleic acids for in vivo genetic manipulation of host bacteria to induce B. phage resistance. Pharmaceutical compositions containing exogenous therapeutic bacteria (e.g., those described herein) can be administered to subjects in need, but in some embodiments, endogenous bacteria in the microbiome of such subjects that are susceptible to one or more target bacteriophages can be genetically engineered in vivo to become resistant to such target bacteriophages, for example, by administering a pharmaceutical composition containing nucleic acid sequences for modulating the resistance of such host bacteria. Accordingly, another embodiment provided herein relates to a pharmaceutical composition containing nucleic acid sequences for modifying the genetic information of host non-pathogenic and symbiotic bacteria in a subject in need, so that such host bacteria are genetically engineered to become resistant to target bacteriophages.

[0116] In some embodiments, the nucleic acid sequence for modifying the genetic information of a host symbiotic bacterium comprises one or more CRISPR spacers targeting one or more target bacteriophages. In some embodiments, the nucleic acid sequence for modifying the genetic information of a host symbiotic bacterium comprises one or more nucleotide sequences encoding one or more Cas polypeptides (e.g., those described herein).

[0117] In some embodiments, the nucleic acid sequence for modifying the genetic information of a host symbiotic bacterium comprises one or more nucleotide sequences encoding one or more variants of bacteriophage receptors on the bacterial cell surface.

[0118] Methods for delivering compositions comprising nucleic acid sequences are known in the art, and those skilled in the art will understand that in some embodiments such nucleic acid sequences may be delivered by recombinant bacteriophages (e.g., as carriers), and in some embodiments such nucleic acid sequences may be delivered by vectors, cosmids, phagemids, or transposons.

[0119] In some embodiments, the nucleic acid sequences according to this disclosure may be delivered by expression vectors. Expression vectors that may be useful for delivering nucleic acid sequences to modulate the resistance of bacterial cells to bacteriophages include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., vectors derived from mouse leukemia virus, splenic necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors.

[0120] In some implementations, the vector may contain one or more transcriptional and / or translational regulatory elements. Depending on the host / vector system used, any of several suitable transcriptional and translational regulatory elements, including constitutive and inductive promoters, transcriptional enhancer elements, and transcriptional terminators, may be used within the expression vector.

[0121] In some embodiments, the vector can be autonomously replicated in a host cell (episomal vector) or integrated into the host cell's genome and replicated together with the host genome (non-episomal mammalian vector). The integrated vector typically contains at least one sequence homologous to the bacterial chromosome, enabling recombination between homologous DNA within the vector and the bacterial chromosome. The integrated vector may also contain bacteriophage or transposon sequences. An episomal vector, or plasmid, is a circular double-stranded DNA loop into which additional DNA segments can be ligated. In some embodiments, plasmids that can be stably maintained within a host are used as expression vectors when recombinant DNA techniques are employed.

[0122] Regulatory sequences include those that direct the constitutive expression of nucleotide sequences and those that direct the inducible expression of nucleotide sequences only under certain environmental conditions. A bacterial promoter is any DNA sequence to which bacterial RNA polymerase can bind and initiate downstream (3') transcription of a coding sequence (e.g., a structural gene) into mRNA. Promoters typically have a transcription initiation region located proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters may also have a second domain called an operator, which may overlap with an adjacent RNA polymerase binding site where RNA synthesis begins. Gene repressor proteins can bind to operators and thereby inhibit the transcription of specific genes; thus, operators enable negatively regulated (inducible) transcription. Constitutive expression can occur in the absence of negative regulatory elements such as operators. In addition, positive regulation may be achieved by gene activation protein binding sequences, which, if present, are typically proximal (5') to the RNA polymerase binding sequence.

[0123] In some embodiments, the nucleic acid sequences according to this disclosure may be delivered by recombinant phages. In some such embodiments, the recombinant phage may be a bacteriophage-derived particle that includes a phagemide, for example, a phagemide containing a nucleic acid sequence (e.g., as described herein) for modulating the resistance of bacterial cells to a target bacteriophage, but does not contain a bacteriophage genome. For example, in some embodiments, the phagemide may include a nucleic acid sequence encoding a CRISPR spacer (e.g., as described herein). Additionally or alternatively, the phagemide may include a nucleic acid sequence encoding a relevant Cas polypeptide.

[0124] The pharmaceutical compositions provided herein may include those suitable for oral administration, including in the cheek and sublingual, intranasal, topical, transdermal, transdermal patch, pulmonary, vaginal, rectal, suppository, mucosal, systemic, or parenteral administration, including intramuscular, intra-arterial, intrathecal, intradermal, intraperitoneal, subcutaneous, and intravenous administration, or those suitable for aerosolization, inhalation, or inhalation.

[0125] In some embodiments, the pharmaceutical compositions described herein may include carriers and excipients (including, but not limited to, buffers, carbohydrates, lipids, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickeners and / or preservatives), metals (e.g., iron, calcium), salts, vitamins, minerals, water, oils (including those of petroleum, animal, plant or synthetic origin), such as peanut oil, soybean oil, mineral oil, sesame oil, physiological saline, aqueous dextrose and glycerol solutions, flavoring agents, colorants, dissociating agents and other acceptable additives, adjuvants, or other pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as binders, pH buffers, isotonic agents, emulsifiers, and wetting agents. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.

[0126] Non-limiting examples of pharmaceutically acceptable excipients suitable for use as described herein include granulators, binders, lubricants, disintegrants, sweeteners, flowing agents, anti-adhesion agents, antistatic agents, surfactants, antioxidants, gums, coatings, colorants, flavoring agents, dispersion enhancers, disintegrants, coatings, plasticizers, preservatives, suspending agents, emulsifiers, plant cellulose materials and spheroidizers, and any combination thereof.

[0127] In some embodiments, the pharmaceutical compositions described herein may be substantially free of preservatives. In some applications, the compositions may contain at least one preservative.

[0128] In some embodiments, the pharmaceutical compositions described herein may be encapsulated in a suitable vehicle, such as liposomes, microspheres, or microparticles. Microspheres formed from polymers or proteins may be designed to pass through the gastrointestinal tract and enter the bloodstream directly. Alternatively, the compounds may be incorporated into microspheres or a complex of microspheres and implanted for slow release over a period ranging from several days to several months.

[0129] In some embodiments, the pharmaceutical compositions described herein can be formulated as sterile solutions or suspensions. Such pharmaceutical compositions may be sterilized by conventional techniques or aseptically filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized. Lyophilized preparations of therapeutic bacteria (e.g., those described herein) may be packaged in a form suitable for oral administration, such as capsules or pills.

[0130] In some embodiments, the pharmaceutical compositions described herein can be administered topically and can be formulated into a variety of topically administered compositions such as solutions, suspensions, lotions, gels, pastes, medicinal sticks, balms, creams, and ointments. Such pharmaceutical compositions may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0131] In some embodiments, the pharmaceutical compositions described herein may be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retaining enemas, containing conventional suppository bases such as cocoa butter or other glycerides, and synthetic polymers such as polyvinylpyrrolidone or PEG. In suppository form of the composition, low-melting-point waxes, such as mixtures of fatty acid glycerides, may optionally be used in combination with cocoa butter.

[0132] When performing the treatment or method of use provided herein, a therapeutically effective amount of the microbial composition (e.g., therapeutic bacteria) and / or pharmaceutical composition described herein is administered to a subject (e.g., a human subject) having a disease, disorder, or condition related to the microbiome dysfunction being treated. The therapeutically effective amount may vary significantly depending on the severity of the disease, the age and relative health status of the subject, the potency of the formulation, and other factors. The subject may be, for example, a human, elderly adult, adult, adolescent, early adolescent, child, infant, or neonatal. The subject may be a patient. The subject may be an individual enrolled in a clinical study. The subject may be an experimental animal, for example, a mammal or rodent.

[0133] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers that facilitate microbial processing into preparations for pharmaceutical use. The formulations can be modified depending on the chosen route of administration. Pharmaceutical compositions described herein can be produced by conventional methods, for example, by conventional mixing, dissolution, granulation, vitrification, spray drying, freeze-drying, dragée production, wet grinding, encapsulation, capture, emulsification, or compression processes.

[0134] In some embodiments, the pharmaceutical composition is prepared in a dry form, for example, by spray drying or freeze-drying. In some embodiments, the pharmaceutical composition is formulated as liquid capsules to maintain the liquid form of the therapeutic bacteria (e.g., those described herein).

[0135] C. Other compositions and formulations The compositions described herein can be formulated for a variety of applications involving the microbiome. In some embodiments, the compositions described herein can be formulated as the pharmaceutical or therapeutic compositions described above. In some embodiments, the compositions described herein may be included in cosmetic compositions. In some embodiments, the compositions described herein may be included in food or beverage products. In some embodiments, the compositions described herein may be included in dietary supplements.

[0136] In some embodiments, compositions containing therapeutic bacteria (e.g., those described herein) may be formulated as nutritional supplements or dietary supplements. For example, in some embodiments, therapeutic bacteria (e.g., those described herein) may be incorporated together with vitamin supplements. In some embodiments, compositions containing therapeutic bacteria (e.g., those described herein) may be formulated in a chewable form, such as probiotic gummies.

[0137] In some embodiments, compositions containing therapeutic bacteria (e.g., those described herein) can be incorporated into the form of foods and / or beverages. Non-limiting examples of foods and beverages into which therapeutic bacteria can be incorporated include, for example, bars, shakes, juices, powdered milk, beverages, frozen foods, fermented foods, and fermented dairy products such as yogurt, yogurt drinks, cheese, lactic acid bacteria drinks, and kefir.

[0138] In some embodiments, compositions containing therapeutic bacteria (e.g., those described herein) can be formulated for use in cosmetics (e.g., skincare or makeup products). Using one or more therapeutic bacteria described herein, cosmetic formulations can be created containing an effective amount of therapeutic bacteria (e.g., those described herein) to treat subjects suffering from or susceptible to skin disorders involving the microbiome. In some embodiments, compositions containing therapeutic bacteria (e.g., those described herein) may be contained in lotions, creams, moisturizers, powders, and the like.

[0139] In some embodiments, the compositions described herein can be administered by a preferred method for delivery to any portion of the gastrointestinal tract of interest, including the oral cavity, mouth, esophagus, stomach, duodenum, small intestinal region including the duodenum, jejunum, ileum, and large intestinal region including the cecum, colon, rectum, and anal canal. In some embodiments, the compositions described herein can be formulated for delivery to the ileum and / or colonic region of the gastrointestinal tract.

[0140] In some embodiments, the compositions described herein may be administered orally, for example, via capsules, pills, powders, tablets, gels, or liquids designed to release such compositions in the gastrointestinal tract. In some embodiments, the compositions described herein may be administered by injection, for example, for formulations containing butyric acid, propionic acid, acetic acid, and / or short-chain fatty acids. In some embodiments, the compositions described herein may be applied to the skin, for example, in the form of creams, liquids, or patches. In some embodiments, the compositions described herein may be administered in the form of suppositories and / or enemas. In some embodiments, a combination of administration routes may be utilized.

[0141] In some embodiments, the compositions described herein may be administered as part of a fecal transplantation process. For example, in some embodiments, such compositions may be administered to a subject by a tube, such as a nasogastric tube, nasojejunal tube, nasoduodenal tube, oral gastric tube, oral jejunal tube, or oral duodenal tube. In some embodiments, the compositions may be administered to a subject by colonoscopy, endoscopy, sigmoidoscopy, and / or enema.

[0142] In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., bacteria described herein) is formulated so that one or more therapeutic bacteria can replicate once delivered to a target habitat (e.g., the intestines). In one non-limiting example, such a bacterial composition is formulated into pills such that the pills have a shelf life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In another non-limiting example, the storage of the bacterial composition is formulated so that the therapeutic bacteria contained therein can replicate while they are in the intestines. In some embodiments, other components may be added to supplement the shelf life of such bacterial composition. In some embodiments, one or more therapeutic bacteria may be formulated in a way that allows them to survive in non-natural environments. For example, bacteria native to the intestines may not survive in oxygen-rich environments. To overcome this limitation, such bacteria may be formulated into pills that can reduce or eliminate exposure to oxygen. Other strategies to extend the shelf life of therapeutic bacteria may involve other microorganisms (for example, if the bacterial community contains a composition in which one or more strains contribute to the survival of one or more strains).

[0143] In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is lyophilized (e.g., freeze-dried) and formulated as a powder, tablet, enteric-coated capsule (e.g., for delivery to the ileum / colon), or pill, which can be administered to a target by any preferred route. Such lyophilized formulations can be mixed with saline or other solutions before administration.

[0144] In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is formulated for oral administration, for example, as an enteric-coated capsule or pill, to deliver the contents of such formulation to the target ileum and / or colon region.

[0145] In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is formulated for oral administration. In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is formulated as enteric-coated pills or capsules for oral administration. In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is formulated for delivery of such therapeutic bacteria to the ileum region of interest. In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is formulated for delivery of such therapeutic bacteria to the colon region of interest (e.g., the upper colon). In some embodiments, a bacterial composition containing therapeutic bacteria (e.g., those described herein) is formulated for delivery of such therapeutic bacteria to the ileum and colon regions of interest.

[0146] In some embodiments, enteric coatings can be used to protect the contents of oral formulations, such as pills or capsules, from stomach acidity and to facilitate delivery to the ileum and / or upper colon region. Non-limiting examples of enteric coatings include pH-sensitive polymers (e.g., eudragit FS30D), methyl acrylate-methacrylate copolymers, cellulose succinate acetate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate acetate (e.g., hypromellose succinate acetate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylate copolymers, shellac, cellulose trimellitate acetate, sodium alginate, zein, other polymers, fatty acids, waxes, shellac, plastics, and plant fibers. In some embodiments, the enteric coating is formed by a pH-sensitive polymer. In some embodiments, the enteric coating is formed by eudragit FS30D.

[0147] In some embodiments, the enteric coating may be designed to dissolve at any preferred pH. In some embodiments, the enteric coating is designed to dissolve at a pH greater than approximately pH 6.5 to approximately pH 7.0. In some embodiments, the enteric coating is designed to dissolve at a pH greater than approximately pH 6.5. In some embodiments, the enteric coating is designed to dissolve at a pH greater than approximately pH 7.0. In some embodiments, the enteric coating may be designed to dissolve at a pH greater than approximately 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5 pH units.

[0148] In some embodiments, the compositions described herein (e.g., pharmaceutical compositions) are formulated to deliver a bacterial population (e.g., as described herein) to the colon. Examples of such formulations include, but are not limited to, pH-sensitive compositions, and more specifically, buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after passing through the stomach. When a pH-sensitive composition is used in the formulation of a pharmaceutical preparation, the pH-sensitive composition is preferably a polymer whose pH threshold for decomposition of the composition is about 6.8 to about 7.5. This numerical range is the range in which the pH shifts to the alkaline side in the distal part of the stomach and is therefore suitable for use in delivery to the colon.

[0149] Another embodiment of a preparation useful for the delivery of a bacterial composition to the colon (e.g., a pharmaceutical preparation) ensures delivery to the colon by delaying the release of the contents (e.g., the bacterial composition) by approximately 3 to 5 hours, corresponding to the small intestinal transit time. In one embodiment of a pharmaceutical preparation for delayed release, a hydrogel is used as a shell. The hydrogel hydrates and swells upon contact with gastrointestinal fluid, resulting in the effective release of the contents (primarily in the colon). The delayed-release dosing unit comprises a drug-containing composition having a material that coats or selectively coats the drug or active ingredient to be administered. Examples of such selective coating materials include in vivo-degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and / or enzymatically degradable polymers. A wide variety of coating materials are available for efficiently delaying release, including, for example, cellulosic polymers such as hydroxypropylcellulose, acrylic polymers and copolymers such as methacrylic polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

[0150] Examples of compositions that enable delivery to the colon include bioadhesive compositions that adhere specifically to the colonic mucosa (e.g., polymers described in U.S. Patent No. 6,368,586), and compositions that incorporate protease inhibitors in particular to protect biopharmaceutical preparations in the gastrointestinal tract from degradation due to protease activity.

[0151] Another example of a system that enables delivery to the colon is a system that delivers a composition to the colon by pressure changes, such as one that uses the pressure changes caused by gas production during bacterial fermentation in the distal part of the stomach to release the contents. Such a system is not particularly limited, and a more specific example is a capsule in which the contents are dispersed in a suppository base and coated with a hydrophobic polymer (e.g., ethylcellulose).

[0152] Another example of a system that enables delivery to the colon is a system for delivering a composition to the colon, which is specifically broken down by enzymes present in the colon (e.g., carbohydrate hydrolases or carbohydrate reductases). Such systems are not limited to those mentioned above, and more specific examples include systems using food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

[0153] In some embodiments, the therapeutic bacteria (e.g., those described herein) are formulated as a population of spores. The spore-containing formulation can be administered by any preferred route described herein. Orally administered spore-containing formulations can survive in the low pH environment of the stomach. The amount of spores used may be, for example, about 1% w / w to about 99% w / w of the total formulation.

[0154] In some embodiments, the formulation comprises one or more genetically modified recombinant bacteria or bacteria. In other embodiments, one or more bacteria are unmodified or non-recombinant. In some embodiments, the formulation comprises bacteria that can be regulated, for example, bacteria containing an operon or promoter for controlling bacterial growth. The bacteria may be produced, grown, or modified using any preferred method, including a recombination method.

[0155] Formulations can be customized for a given subject. Custom formulations may include, for example, prebiotics, probiotics, antibiotics, or combinations of active ingredients as described herein. For example, subject-specific data, including age, sex, and weight, can be combined with analysis results to provide a therapeutic agent tailored to the subject. For instance, a subject's microbiome found to be elevated in a particular bacteriophage compared to a healthy subpopulation matched for age and sex may be provided with a therapeutic and / or cosmetic formulation containing an isolated or concentrated population of phage-resistant bacteria targeting the identified bacteriophage.

[0156] The compositions provided herein can be stored at any suitable temperature. Formulations can be stored refrigerated at temperatures such as about -80°C, about -20°C, about -4°C, or about 4°C. In some embodiments, formulations can be prepared for storage temperatures of about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 12°C, about 14°C, about 16°C, about 20°C, about 22°C, or about 25°C. In some embodiments, the storage temperature is about 2°C to about 8°C. In some embodiments, storage of microbial compositions (e.g., including therapeutic bacteria described herein) at low temperatures, such as about 2°C to about 8°C, can allow the microorganisms to survive and increase the efficiency of the composition, for example, when present in a liquid or gel formulation. In some embodiments, stability can be further extended by storage at a freezing temperature below 0°C with a cryoprotectant.

[0157] The pH of the compositions described herein may range from about 3 to about 12, depending on the application (e.g., intestinal delivery vs. skin delivery). The pH of such compositions may be, for example, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 11, or about 11 to about 12 pH units. The pH of the compositions may be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the compositions may be, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 pH units. The pH of the compositions may be, for example, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, or up to 12 pH units. If the pH is outside the range desired by the formulation, the pH can be adjusted by using a sufficient amount of pharmaceutically acceptable acids and bases. In some embodiments, the pH of the composition is about 4 to about 6.

[0158] D. Optional additives In some embodiments, the compositions described herein may include prebiotics. In some embodiments, the prebiotics may be inulin or may include inulin. Inulin can serve as an energy source for the bacterial formulation.

[0159] In some embodiments, the compositions described herein may contain one or more active agents or therapeutic agents. Exemplary active agents or therapeutic agents may include, but are not limited to, antibiotics, prebiotics, probiotics, glycans (e.g., as decoys that can restrict specific bacterial / viral binding to the intestinal wall), bacteriophages, microorganisms, and the like.

[0160] In some embodiments, the compositions described herein may contain one or more active ingredients for enhancing the stability and / or viability of bacterial formulations. Non-limiting examples of such stabilizers include gene elements, glycerin, ascorbic acid, skim milk, lactose, tween®, alginates, xanthan gum, carrageenan gum, mannitol, palm oil, poly-L-lysine (POPL), and combinations thereof.

[0161] E. Exemplary Dosage Forms The appropriate amount of the pharmaceutical composition administered, the number of treatments, and the unit dose may vary depending on the subject and / or the disease state of the subject.

[0162] The pharmaceutical compositions described herein may be in unit dosage forms suitable for single-dose administration of precise doses. In unit dosage forms, the pharmaceutical formulations described herein may be divided into unit doses containing appropriate amounts of one or more microbial compositions. Unit doses may be in the form of packages containing separate amounts of the formulation. Non-limiting examples include liquids in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose, non-resealable containers. Compositions may be in multi-dose formats. Multi-dose, resealable containers may be used, for example, in combination with preservatives. Formulations for parenteral injection may be presented in unit dosage forms, e.g., ampoules, or multi-dose containers containing preservatives.

[0163] In some embodiments, the dosage may be in the form of a solid, semi-solid, or liquid composition. Non-limiting examples of dosage forms suitable for use according to this disclosure include feeds, foods, pellets, licks, liquids, elixirs, aerosols, inhalants, sprays, powders, tablets, pills, capsules, gels, gel tabs, nanosuspensions, nanoparticles, microgels, suppositories, lozenges, aqueous or oily suspensions, ointments, patches, lotions, toothpastes, emulsions, creams, drops, dispersible powders or granules, emulsions in hard or soft gel capsules, syrups, herbal medicines, nutritional supplements, dietary supplements, and any combination thereof.

[0164] Therapeutic bacteria (e.g., those described herein) can be present in a pharmaceutically suitable concentration in a pharmaceutical composition. The concentration of the therapeutic bacteria can be, for example, about 10 1 to about 10 18 colony forming units (CFU). The concentration of the therapeutic bacteria (e.g., those described herein) can be, for example, at least 10 1 , at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , or at least 10 18 CFU. The concentration of the therapeutic bacteria (e.g., those described herein) can be, for example, up to 10 1 , up to 10 2 , up to 10 3 , up to 10 4 , up to 10 5 , up to 10 6 , up to 10 7 , up to 10 8 , up to 10 9 , up to 10 10 , up to 10 11 , up to 10 12 , up to 10 13 , up to 10 14 , up to 10 15 , up to 10 16 [[ID=7上一句: 17 17 , or up to 10 18 CFU. In some embodiments, the concentration of the therapeutic bacteria (e.g., those described herein) is about 10 8 CFU to about 109 It is CFU.

[0165] The pharmaceutical compositions described herein can be formulated with prebiotics at suitable therapeutic concentrations. For example, therapeutic concentrations of prebiotics may be at least about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 125 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. For example, the therapeutically effective concentration of prebiotics may be approximately 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 110 mg / ml, 125 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml. For example, the therapeutically effective concentration of prebiotics may be approximately 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 110 mg / ml, 125 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml.

[0166] In some embodiments, the pharmaceutical compositions described herein are administered, for example, once, twice, three, four, five times, or more times per day. In some embodiments, the pharmaceutical compositions described herein may be administered, for example, daily, every other day, three times a week, twice a week, once a week, or at other appropriate intervals for the treatment of a condition.

[0167] F. Kit The compositions described herein may be packaged as kits. In some embodiments, the kit includes written instructions for the administration / use of the compositions. The written materials may be, for example, labels. The written materials may suggest conditions and methods of administration. The instructions provide the subject and supervising physician with the best guidance to achieve optimal clinical outcomes from the administration of the treatment. The written materials may be labels. In some embodiments, the labels may be approved by a regulatory body, for example, the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or other regulatory bodies.

[0168] III. Exemplary Use The therapeutic bacteria, compositions, and methods provided herein may be useful for the treatment and / or prevention of diseases, disorders, or conditions associated with microbiome dysfunction. Therefore, the technologies provided herein are suitable for subjects suffering from or susceptible to diseases, disorders, or conditions associated with microbiome dysfunction. In some embodiments, the technologies provided herein are suitable for subjects suffering from or susceptible to diseases, disorders, or conditions associated with intestinal microbiome dysfunction.

[0169] In some embodiments, a method is provided which involves exposing an object suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction to a population of therapeutic bacteria (e.g., those described herein) or a composition described herein (e.g., including a pharmaceutical composition, a cosmetic composition, a food or beverage, or a nutritional supplement).

[0170] In some embodiments, the therapeutic bacteria are exposed to a target that needs them (e.g., a target suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction), and each includes clustered, regularly spaced, short-palindrical repeat (CRISPR) spacers targeting one or more bacteriophages. In some embodiments, the therapeutic bacteria are exposed to a target that needs them (e.g., a target suffering from or susceptible to a disease, disorder, or condition associated with microbiome dysfunction), and each includes at least one variant of a bacteriophage receptor on the bacterial cell surface.

[0171] In some embodiments, such a population of therapeutic bacteria is exposed to subjects suffering from or susceptible to diseases, disorders, or conditions associated with gut microbiome dysfunction. Examples of diseases, disorders, or conditions associated with gut microbiome dysfunction include, but are not limited to, inflammatory bowel disease (IBD) or irritable bowel syndrome, Crohn's disease, ulcerative colitis, and immunotherapy-associated colitis. In some such embodiments, the therapeutic bacteria exposed to subjects suffering from or susceptible to diseases, disorders, or conditions associated with gut microbiome dysfunction may be caudovirales or resistant to one or more bacteriophages that may include them.

[0172] In some embodiments, the step of exposing a subject in need to a population of therapeutic bacteria (e.g., as described herein) includes administering a composition containing the population of therapeutic bacteria (e.g., as described herein) to such subject.

[0173] In some embodiments, the step of exposing a subject in need to a population of therapeutic bacteria (e.g., as described herein) includes administering to such subject a composition comprising a nucleic acid sequence for modifying the genome of a host symbiont in the subject so that the host symbiont becomes resistant to a target bacteriophage. In some embodiments, the nucleic acid sequence for modifying the genome of a host symbiont comprises one or more CRISPR spacers targeting one or more target bacteriophages.

[0174] In some embodiments, the step of exposing a subject in need to a population of therapeutic bacteria (e.g., those described herein) includes administering to such subject one of the compositions described herein (e.g., pharmaceutical compositions, cosmetic compositions, foods or beverages, or dietary supplements).

[0175] In some embodiments, the therapeutic bacterial populations or compositions described herein (e.g., including pharmaceutical compositions, cosmetic compositions, foods or beverages, or nutritional supplements) are administered before, during, and / or after treatment with antimicrobial agents such as antibiotics. For example, in some embodiments, the therapeutic bacterial populations or compositions described herein may be administered at least 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year prior to and / or after antibiotic treatment. In some embodiments, the therapeutic bacterial populations or compositions described herein may be administered up to 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year prior to and / or after antibiotic treatment.

[0176] In some embodiments, the therapeutic bacterial populations or compositions described herein (e.g., including pharmaceutical compositions, cosmetic compositions, foods or beverages, or dietary supplements) are administered after antibiotic treatment. For example, in some embodiments, the therapeutic bacterial populations or compositions described herein may be administered after the completion of an antibiotic regimen or the entire course of treatment.

[0177] In some embodiments, the group or composition of therapeutic bacteria described herein (including, for example, a pharmaceutical composition, a food or beverage, or a nutritional supplement) is administered to the subject before, during, and / or after food intake. In some embodiments, the group or composition of therapeutic bacteria described herein is administered together with food intake. In some embodiments, the group or composition of therapeutic bacteria described herein is administered together with (for example, simultaneously with) food intake.

[0178] In some embodiments, the therapeutic bacterial populations or compositions described herein (e.g., including pharmaceutical compositions, foods or beverages, or dietary supplements) are administered to the subject before food intake. In some embodiments, the therapeutic bacterial populations or compositions described herein may be more effective or potent in treating bacterial conditions (e.g., bacterial conditions associated with microbiome dysfunction) when administered before food intake. For example, in some embodiments, the therapeutic bacterial populations or compositions described herein may be administered to the subject at least about 1 minute, about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, or about 1 day before food intake. In some embodiments, a population or composition of therapeutic bacteria described herein may be administered by a subject up to about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, or 1 day prior to food ingestion.

[0179] In some embodiments, the therapeutic bacterial populations or compositions described herein (including, for example, pharmaceutical compositions, foods or beverages, or dietary supplements) are administered after food ingestion by the subject. In some embodiments, the therapeutic bacterial populations or compositions described herein are more effective or potent in treating bacterial conditions (e.g., bacterial conditions associated with microbiome dysfunction) when administered after food ingestion. For example, in some embodiments, the therapeutic bacterial populations or compositions described herein may be administered at least about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after food ingestion by the subject. In some embodiments, the therapeutic bacterial populations or compositions described herein may be administered up to about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after food ingestion by the subject.

[0180] Multiple genera or species of therapeutic bacteria can be administered in any order or simultaneously. When administered simultaneously, multiple genera or species of therapeutic bacteria may be provided in a single unified form or in multiple forms, for example, as multiple separate pills. In some embodiments, multiple genera or species of therapeutic bacteria may be packaged together or separately in a single package or in multiple packages. In some embodiments, one or all of the genera or species of therapeutic bacteria may be given in multiple doses. If not administered simultaneously, the timing between multiple doses may vary up to about one month.

[0181] The compositions described herein may be administered before, during, or after the onset of a disease or condition associated with microbiome dysfunction, and the timing of administration of such compositions may vary. For example, in some embodiments, the compositions described herein may be used as prophylactic agents and may be continuously administered to subjects prone to conditions or diseases associated with microbiome dysfunction in order to reduce the likelihood of such disease or condition occurring. In some embodiments, the compositions described herein may be administered to subjects during or as soon as possible after the onset of symptoms. In some embodiments, administration of the compositions described herein (e.g., including therapeutic bacteria) may be initiated within the first 48 hours of the onset of one or more symptoms, within the first 24 hours of the onset of one or more symptoms, within the first 6 hours of the onset of one or more symptoms, or within 3 hours of the onset of one or more symptoms. In some embodiments, the initial dose may be carried out using the compositions described herein via any route, such as any route described herein. In some embodiments, the compositions described herein (e.g., including therapeutic bacteria) may be administered as soon as feasible, after the onset of a disease or condition associated with microbiome dysfunction is detected or suspected, and for a period of time appropriate for treating such disease or condition, e.g., about one to three months. The duration of treatment may vary depending on the subject.

[0182] The compositions and / or methods described herein may be useful for the treatment and / or prevention of diseases, disorders, or conditions associated with microbiome dysfunction. In some embodiments, the compositions and / or methods described herein are generally applicable to animals, particularly humans and economically viable livestock.

[0183] In some embodiments, the diseases, disorders, or conditions related to microbiome dysfunction for which the compositions and / or methods described herein can be utilized are chronic disorders associated with the presence of an abnormal gut microbiota. Such disorders include, but are not limited to, conditions belonging to the following categories: - Gastrointestinal disorders including irritable bowel syndrome or spastic colon, functional bowel disorders (FBD) including constipation-predominant FBD, pain-predominant FBD, and epigastric FBD, non-ulcerative dyspepsia (NUD), gastroesophageal reflux, inflammatory bowel disease including Crohn's disease, ulcerative colitis, atypical colitis, collagen colitis, microscopic colitis, chronic Clostridium difficile infection, pseudomembranous colitis, mucosal colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticular disease, AIDS intestinal disorders, small intestinal bacterial overload, celiac disease, colonic polyposis, colonic polyps, chronic idiopathic pseudo-obstructive syndrome; - Chronic intestinal infections caused by certain pathogens, including bacteria, viruses, fungi, and protozoa; - Viral gastrointestinal disorders, including viral gastroenteritis, Norwalk virus gastroenteritis, rotavirus gastroenteritis, and AIDS-associated gastroenteritis; - Liver disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver, or latent cirrhosis; - Rheumatic diseases such as rheumatoid arthritis, non-rheumatoid arthritis, non-rheumatic factor-positive arthritis, ankylosing spondylitis, Lyme disease, and Reiter's syndrome; - Immune-mediated disorders such as glomerulonephritis, hemolytic urea syndrome, juvenile diabetes mellitus, mixed frozen glomerulonephritis, polyarteritis, familial Mediterranean fever, amyloidosis, scleroderma, systemic lupus erythematosus, and Behçet's syndrome; - Autoimmune disorders, including systemic lupus, idiopathic thrombocytopenic purpura, Sjögren's syndrome, hemolytic uremic syndrome, or scleroderma; - Neurological syndromes such as chronic fatigue syndrome, migraines, multiple sclerosis, amyotrophic lateral sclerosis, myasthenia gravis, Guillain-Barré syndrome, Parkinson's disease, Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, and other degenerative disorders; - Mental disorders, including chronic depression, schizophrenia, psychotic disorders, and bipolar disorder; - Regressive disorders, including Asperger's syndrome, Rett syndrome, attention deficit hyperactivity disorder (ADHD), and attention deficit disorder (ADD); - Degenerative disorders, autism; - Sudden Infant Death Syndrome (SIDS), Anorexia Nervosa; - Skin diseases such as chronic urticaria, acne, herpetic dermatitis, and vascular disorders.

[0184] In some embodiments, the diseases, disorders, or conditions associated with microbiome dysfunction for which the compositions and / or methods described herein can be utilized are chronic disorders associated with the presence of abnormalities or abnormal distributions of the microbiome in the gastrointestinal tract of a mammalian host. Exemplary diseases, disorders, or conditions associated with intestinal microbiome dysfunction include, but are not limited to, inflammatory bowel disease (IBD) or irritable bowel syndrome, Crohn's disease, ulcerative colitis, and immune-associated colitis (e.g., immunotherapy patients who have developed colitis). In some embodiments, the subjects to whom the therapeutic bacteria described herein are administered may be those who have previously received probiotic therapy (e.g., including patients who have experienced or are suffering from probiotic therapy failure), fecal microbiota transplantation (FMT) (e.g., including patients who have experienced or are suffering from FMT failure), and / or immunotherapy (e.g., colitis-associated immunotherapy).

[0185] In some embodiments, the compositions described herein may be administered in conjunction with other treatments, including, for example, antibiotic therapy, immunotherapy, chemotherapy, radiotherapy, anti-inflammatory agents, antiviral agents, antimicrobial agents, antifungal agents, probiotic therapy, fecal microbiota transplantation, and combinations thereof. In some embodiments, the compositions described herein may be administered before another treatment (e.g., those described herein). In some embodiments, the compositions described herein may be administered after another treatment (e.g., those described herein). For example, in some embodiments, a short course of antibiotics may be administered before treatment with the compositions described herein, for example, to eliminate tissue-invasive pathogens originating in the intestinal lumen. For example, in the treatment of Crohn's disease, in some embodiments, anti-tuberculosis therapy may be required 6 to 12 weeks before administration of the compositions described herein, as a result of clearing the intestines and replacing the microbiota contents with a predetermined microbiota.

[0186] In some embodiments, prior to administering the compositions described herein, colon cleansing methods such as colon perfusion / hydrotherapy, antibiotic therapy, enema, administration of laxatives, nutritional supplements, dietary fiber, enzymes, and magnesium may be performed. [Examples]

[0187] Example 1. In vitro co-culture of microbiome phages and bacteria As described herein, this disclosure encompasses the insight that bacteriophages (or phages) may be present in certain human organs or tissues (e.g., the gut) that deplete beneficial bacterial populations. In light of this insight, phage fractions were isolated from fecal samples of healthy individuals and added to bacterial cultures derived from the gut microbiome of the same individuals. 16s rRNA sequencing was then performed to characterize the changes in the composition of the bacterial community after phage addition. See, for example, Figures 1A–1B. The inventors found that phage addition resulted in the depletion of non-pathogenic commensal bacteria known to be important for maintaining health. In particular, Bifidobacterium longum and Clostridium scindens were found to be depleted after phage addition (Figures 2B–2C). The identification of phages capable of depleting such bacteria surprisingly confirms the insight provided by this disclosure that phages may drive dysbiosis and directly infect and deplete probiotic therapies. This method can be adapted, for example, to determine the in vivo impact of phages on beneficial bacterial populations by isolating phage fractions from individual fecal samples, adding them to mice colonized with cultured bacteria derived from the gut microbiome of the same individual or other individuals, and characterizing the phage's impact on the bacterial community.

[0188] Furthermore, it was found that phages that infect Clostridia are more common in IBD patients than in healthy populations (Figure 4).

[0189] The following is an exemplary list of Clostridia bacteria whose phages have been identified to infect these species / strains present in the intestines of patients with IBD. [Table 1]

[0190] Example method: Fecal samples were homogenized in 12 ml of sterile 20% glycerol / 1×PBS in a 50 ml cone, and serial dilutions were plated onto 0.1% mucin BHI agar. The plates were scraped and diluted in PBS. 5 µl of the diluted sample was added to 5 ml of 0.1% BHI medium. 2 µl of virus-like particles (VLPs) isolated from the same sample were added to the plate culture. E. coli and T7 phage were added to a subset of the culture as positive controls. The cultures were grown anaerobically for 72 hours. The samples were then spun down and prepared for sequencing.

[0191] Example 2. A computational approach to identify phages and their bacterial hosts present in patients with inflammatory bowel disease. Using a CRISPR-based approach, it is possible to predict the bacterial targets or hosts of phages present throughout an individual. A curated list of CRISPR spacer sequences extracted from a wide range of gut bacteria was analyzed. Gregory et al. “The human gut virome database” bioRxiv 655910 (May 2019). The presence of a given CRISPR spacer sequence in a bacterial population provides evidence that a phage containing that sequence has infected that bacterium. Furthermore, phages present in individuals that attack putative non-pathogenic symbiotic bacterial hosts and / or non-pathogenic symbiotic bacteria can be identified by matching viral sequences to CRISPR spacer sequences from known bacterial hosts. See, for example, Figure 3. Using such information, phage-resistant non-pathogenic symbiotic bacteria can be developed.

[0192] Example 3. Manipulation of exemplary phage-resistant non-pathogenic symbiotic bacteria Phage-resistant, non-pathogenic symbiotic bacteria engineered to block infectious enterophages in vitro.

[0193] As described in Examples 1 and 2, the data presented herein suggest that infectious phages (e.g., those present in the gut) deplete bacteria in the microbiome, and that phages targeting beneficial bacteria are significantly more common in patients with IBD. Based on these findings, commensal bacteria are engineered to be resistant to infectious phages targeting gut bacteria. The effectiveness of these bacteria in resisting phage proliferation in vitro and in vivo is then evaluated.

[0194] Phage-resistant bacteria are manipulated based on the identification of a bacterial host for the phage and corresponding phage sequence information. In some embodiments, one or more sequences matching one or more identified phages are introduced into the CRISPR (e.g., CRISPR-Cas) locus of a non-pathogenic symbiotic bacterial strain. Methods for modulating intracellular resistance to target nucleic acids are known in the art. See, for example, U.S. Patent No. 10,066,233. Therefore, those skilled in the art will understand, upon reading this disclosure, that such methods and other methods known in the art (e.g., CRISPR-Cas technology) may be useful for manipulating therapeutic compositions of non-pathogenic and symbiotic bacteria that are resistant to one or more bacteriophages (e.g., as described herein). In some embodiments, bacteriophage-resistant non-pathogenic and symbiotic bacteria can be manipulated by inserting one or more target phage sequences as CRISPR spacers within a CRISPR array (e.g., an endogenous CRISPR array or an exogenous / heterogeneous CRISPR array) present in the non-pathogenic and symbiotic bacterial strain of interest. For example, in some embodiments, phages that infect a given non-pathogenic and symbiotic bacterial strain of interest can be identified using the techniques described in Examples 1 and 2. In some embodiments, CRISPR-Cas locus sequences in non-pathogenic and symbiotic bacterial strains can be identified by sequencing relevant portions of the bacterial strain's genome (e.g., the entire genome in some embodiments). In some such embodiments, protospacer-adjacent motif (PAM) sequences recognized by the identified CRISPR-Cas system are characterized. See, for example, Mendoza and Trinh, Biotechnol J. (2018) 13:e1700595, and Gleditzsch et al., RNA Biol. (2019) 16:504-517. A suitable spacer sequence is typically selected from the genome of a phage identified to infect a given non-pathogenic and symbiotic bacterial strain of interest. In some embodiments, the suitable spacer sequence is or includes a conserved sequence.In some embodiments, such conserved sequences may be present in phage genes (e.g., phage genes present in phages of different genera, species, and / or strains). In some embodiments, a preferred spacer sequence may be, or include, a sequence of up to eight nucleotides upstream of a PAM sequence. A CRISPR unit containing, or consisting of, a preferred spacer sequence adjacent to two repeat elements (e.g., a CRISPR repeat) is then introduced into the non-pathogenic and symbiotic bacterial strains of interest. In some embodiments, the spacer sequence can be introduced into an endogenous CRISPR-Cas array of the non-pathogenic and symbiotic bacteria. In some embodiments, the spacer sequence(s) may be introduced in trans onto a synthetic array (e.g., on the genome or elsewhere on the plasmid) that is compatible with the endogenous Cas mechanism. In some embodiments, the spacer sequence(s) may be introduced in trans into a synthetic array that is co-delivered with an exogenous Cas gene.

[0195] In some embodiments, multiple spacer sequences can be used to target distinct phage populations. Therefore, in some embodiments, at least one (e.g., including at least two) spacer sequences targeting a single bacteriophage population are introduced into the CRISPR locus of a non-pathogenic symbiotic bacterium. Spacer diversity can enhance protection against phage infection. Thus, in some such embodiments, at least two spacer sequences are identical, while in some such embodiments, at least two spacer sequences are distinct.

[0196] In some embodiments, multiple (e.g., at least two, at least three, or more) spacer sequences targeting different phage populations or families can be introduced into a single non-pathogenic and symbiotic bacterium. Such manipulated non-pathogenic and symbiotic bacteria may be useful in providing resistance to multiple different phage populations or families.

[0197] In some embodiments, the spacer sequence introduced into the CRISPR locus of a non-pathogenic symbiotic bacterium is 20–50 bp long. In some embodiments, the spacer sequence introduced into the CRISPR locus of a non-pathogenic symbiotic bacterium (e.g., the CRISPR-Cas locus) is selected from those identified to match CRISPR spacer sequences identified in a given bacterial host, and / or can also be selected from phage sequences. In some embodiments, sequences that are conserved among various phage populations (or across entire populations) are selected.

[0198] In some embodiments, the non-pathogenic symbiotic bacteria described herein may play an important role in maintaining health and / or may be useful as therapeutic agents for treating certain chronic conditions.

[0199] Tools for genetic manipulation are well known in the art, and it will be understood that such tools can be useful for manipulating non-pathogenic symbiotic bacteria to become phage-resistant as described herein. As will be understood by those skilled in the art, in some embodiments, phage-resistant non-pathogenic symbiotic bacteria may be manipulated using CRISPR / Cas9-based genome editing tools that have the advantage of efficient markerless gene editing. For example, for information on methods of CRISPR-Cas identification and / or characterization, spacer selection and / or manipulation, and / or genome editing using the CRISPR-Cas system, see Barrangou et al. Science (2007) 315:1709-1712, Deveau et al. Journal of Bacteriology (2008) 190:1390-1400, Barrangou and Marraffini, Mol Cell (2014) 54:234-244, Crawley et al. CRISPR J. (2018) 1:171-181, Crawley et al. Scientific Reports (2018) 8:11544, and Hidalgo-Cantabrana et al. PNAS (2019) 116:15774-15783. The contents of each reference cited herein are incorporated herein by reference in their entirety for the purposes described herein.

[0200] Engineered phage-resistant non-pathogenic commensal bacteria can be evaluated in vitro using methods known in the art. For example, in some embodiments, engineered phage-resistant non-pathogenic commensal bacteria can be evaluated in vitro by measuring the survival of such engineered bacteria in the presence of infectious phages. Infectious phages can be collected from the viral fraction of a fecal sample from a patient in whom infectious phages have been identified. The collected viral fraction is then filtered and sterilized. The phages from the filtered viral fraction are added to a culture (e.g., monoculture) of (i) one or more engineered phage-resistant non-pathogenic commensal bacterial strains, or (ii) a wild-type (WT) strain as a control. The growth of the bacterial strain in the presence of phages is evaluated, for example, using a plaque assay.

[0201] Growth in monocultures may differ from growth in mixed bacterial cultures of bacteria, such as gut-derived microbiomes. Therefore, in some embodiments, the survival of manipulated phage-resistant non-pathogenic symbiotic bacteria can be evaluated in mixed bacterial cultures in the presence of infectious phages.

[0202] Example 4. Manipulating exemplary phage-resistant symbiotic bacteria in vivo. A human microbiome-associated (HMA) mouse model is used to evaluate the ability of engineered phage-resistant bacteria to resist phage predation and promote colonization in vivo. Antibiotic-pretreated mice are colonized with fecal samples in which infectious phages are identified against a target non-pathogenic commensal bacterial host, resulting in robust colonization of mice with a human microbiome. Engineered phage-resistant non-pathogenic commensal bacteria (e.g., those demonstrating robust efficacy in an in vitro assay, as described in Example 3 above) or WT strains are then added to the HMA mice. The overall microbiome and the composition of the engineered phage-resistant non-pathogenic commensal bacterial strains can be monitored weekly via 16S rRNA sequencing and qPCR over a set period (e.g., 28 days).

[0203] In some embodiments, the engineered phage-resistant bacteria are evaluated in a specific pathogen-free (SPF) mouse model. For example, a therapeutic composition comprising (i) a phage targeting the non-pathogenic and symbiotic bacteria of interest, and (ii) phage-resistant non-pathogenic and symbiotic bacteria (e.g., as described herein), is introduced into the intestines of SPF mice. The levels of the phage and phage-resistant bacterial strains are evaluated, for example, by qPCR using primers complementary to the engineered bacterial strains.

[0204] Phages will also be isolated from mouse fecal samples and sequenced on these samples to evaluate whether the replication of target phages is reduced in the presence of phage-resistant, non-pathogenic symbiotic bacterial strains.

[0205] Example 5. Using exemplary phage-resistant Clostridium to treat inflammatory bowel disease (IBD). As discussed above, the inventors have found that phages that attack Clostridium class bacteria are significantly more common in patients with IBD. Such Clostridium bacteria primarily include several species that have been shown to have anti-inflammatory effects. For example, many of these Clostridium species have been previously reported to promote the induction of colonic regulatory T (Treg) cells in mice and the oral administration of a mixture of 17 Clostridium strains that attenuated the disease in a mouse model of colitis. These findings suggest that phage-driven reduction of anti-inflammatory Clostridium species may promote or contribute to the development of inflammation and IBD disease in susceptible individuals.

[0206] In a mouse model of IBD, bacterial and viral fractions are isolated from fecal samples from healthy individuals or IBD patients to evaluate the ability of phage-resistant bacteria to treat dysbiosis and disease. Mice pre-treated with antibiotics are then colonized with the bacterial fraction from healthy individuals, for example, by forced oral administration. The mice are then orally inoculated with phages isolated from healthy individuals or individuals with IBD, and one or more phage-resistant or WT non-pathogenic commensal bacterial strains. The levels of phage-resistant and WT bacteria are tracked over time, for example, using qPCR, and disease status can be monitored via body weight, latent blood measurement, and / or inflammatory markers.

[0207] The effectiveness of phage-resistant non-pathogenic commensal bacteria can also be evaluated using mice that colonize directly in fecal samples from one or more individuals with IBD. WT and phage-resistant non-pathogenic commensal bacteria may be administered, and the levels of such bacteria may be tracked over time, followed by monitoring of the disease state via body weight, latent blood measurements, and / or inflammatory markers.

[0208] Example 6. Manipulating phage-resistant Bifidobacterium longum In some embodiments, Bifidobacterium longum present in the human gut can possess an IC-type CRISPR system. In some embodiments, resistance can be conferred to these CRISPR arrays by introducing spacer sequences that match a target phage present in the gut that depletes these bacteria. Phage-resistant bacteria can be evaluated for improved growth in the presence of the target phage in vitro and in vivo.

[0209] Example 7. Manipulating phage-resistant Lactobacillus Several symbiotic species of Lactobacillus have been shown to be important for human health, including, but not limited to, Lactobacillus gasseri, Lactobacillus crispatus, and Lactobacillus acidophilus. In some embodiments, phage-resistant strains of Lactobacillus can be engineered to resist phages present in the human gut microbiome using the method described in Example 3. Therapeutic compositions containing such engineered phage-resistant strains may be useful for administration to affected individuals to treat diseases associated with microbiome dysfunction.

[0210] Equivalents and Scope In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or otherwise evident from the context. A claim or statement containing “or” between one or more members of a group is deemed to apply unless otherwise stated or otherwise evident from the context, if one, two or more, or all of the members of that group are present in, used in, or otherwise related to a given product or process. The present invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise related to a given product or process. The present invention includes embodiments in which two or more, or all, of the members of a group are present in, used in, or otherwise related to a given product or process.

[0211] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more of the enumerated claims are introduced in another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented enumerated, for example, in Markush group form, each subgroup of the element is also disclosed, and any element(s) may be removed from the group. Generally, where the present invention or an aspect of the present invention is considered to include certain elements and / or features, it should be understood that certain embodiments of the present invention or aspects of the present invention consist of, or essentially consist of, such elements and / or features. For brevity, those embodiments are not specifically described in this specification in these terms. Note that the terms “comprising” and “containing” are intended to be open and allow for the inclusion of additional elements or steps. Where a scope is given, the endpoints are included. Furthermore, unless otherwise specified, or unless otherwise evident from the context and the understanding of those skilled in the art, values ​​expressed as a range may refer to any specific value or subrange within the ranges expressed in different embodiments of the invention, up to one-tenth of the lower limit unit of the range, unless the context clearly indicates otherwise.

[0212] Those skilled in the art will be able to understand, or verify by using merely routine experiments, many equivalents of the specific embodiments of the Invention described herein. Unless otherwise stated, or unless it is obvious to those skilled in the art that a contradiction or inconsistency would occur, the Invention should be understood to encompass all variations, combinations, and permutations introduced into one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims into another claim (or any other related claim) dependent on the same basic claim. Furthermore, it should be understood that any embodiment or aspect of the Invention may be expressly excluded from the claims, whether or not a particular exclusion is described herein. The scope of the Invention is not intended to be limited to the detailed description of the Invention above, but rather as set forth in the following claims.

Claims

[Claim 1] The invention described in the specification.

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