Microbiota restoration therapy (MRT) compositions and methods of administration

Abstract

Disclosed herein are methods for administering a dose of a microbiota restoration therapy composition by colonoscopy. The methods disclosed herein are useful for treating diseases and / or conditions of the digestive tract, such as Clostridioides difficile infection (GDI), including recurrent GDI, and other gastrointestinal disorders. The method may result in improved quality of life. The methods may alter gut microbiota composition and / or gut metabolome composition. The microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

Description

Atty. Docket No. 052209-0664MICROBIOTA RESTORATION THERAPY (MRT) COMPOSITIONS AND METHODS OF ADMINISTRATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 536,756, filed September 6, 2023, the entire contents of which is incorporated by reference herein.FIELD

[0002] The present disclosure pertains to compositions and methods for administering a microbiota restoration therapy composition by colonoscopy or rectally. In some aspects, the methods are for treating diseases and / or conditions of the digestive tract, such as for treating Clostridioides difficile infection (CDI), including recurrent CDI, by administering a microbiota restoration therapy composition by colonoscopy. In other aspects, the methods are for treating other gastrointestinal diseases or disorders, such as ulcerative colitis (UC) and Crohn’s disease (CD). In other aspects, the methods are for treating cancer. Also disclosed are methods and uses of microbiota restoration therapy compositions as described herein to decolonize patients that carry antibiotic resistance genes (ARGs), e.g., in their gut microbiota, to achieve antimicrobial resistance (AMR) gut decolonization, e.g, to reduce antimicrobial resistance gene abundance and / or reduce the presence of antimicrobial resistant organisms. Also disclosed are methods and uses of microbiota restoration therapy compositions as described herein for altering the gut microbiota composition and / or altering the gut metabolome composition.BACKGROUND

[0003] More than 1000 different species of bacteria reside in a healthy gastrointestinal (GI) tract. Clostridia are anaerobic, spore-forming bacteria. Certain species of Clostridia are pathogens, producing toxins that can be harmful to humans. Clostridioides difficile (also known as Clostridium difficile, hereafter denoted “C. difficile”') is one species of Clostridium that, if overpopulated in the GI tract, can release toxins that can cause a number of symptoms, includingAtty. Docket No. 052209-0664 bloating, constipation, diarrhea, inflammation, abdominal pain, among others that, in certain cases, can lead to death.

[0004] C. difficile infection (CDI) is a common healthcare-associated infection and is commonly acquired in community settings such as nursing homes. CDI contributes to considerable morbidity, mortality, and healthcare expenditure. A major complication from CDI is recurrent infection despite absence of additional risk factors. In recent years, CDI has demonstrated an escalation in overall prevalence and severity of disease. A study of data from 2017 reported that C. difficile accounted for nearly half a million infections in the United States (US). A major complication from CDI is recurrent infection, despite absence of additional risk factors.

[0005] While mature colonic microbiota in healthy adults are generally resistant to C. difficile colonization, a widely accepted model for CDI pathogenesis relates to the use of broad-spectrum antimicrobials which suppress and disrupt microbial communities that normally prevent expansion of C. difficile. Because C. difficile spores are largely resistant to antibiotics, they can germinate into vegetative forms after antibiotic treatment has been discontinued. If residual normal intestinal microbiota cannot restrain the infection, C. difficile bacteria proliferate and produce toxins that cause destruction of colonic epithelial cells, inflammation, and disease symptoms.

[0006] Conventional treatments for CDI utilize antibiotics, which further disrupt the gut bacteria flora and limit recovery of the gut microbiota following CDI. This decreased diversity of gut microbiota following antibiotic treatment can predispose patients to re-colonization with C. difficile and lead to recurrent infections (e.g., recurrent CDI / rCDI). Repeated bouts of CDI can continue for years, leading to persistent use of antibiotics, markedly reduced quality of life (QoL), repeated hospitalizations, and even death. Thus, there exists a need for more effective treatments for treating CDI, including recurrent CDI (rCDI).

[0007] The disruption of gut microbiota has been implicated in other diseases and conditions of the digestive tract and other gastrointestinal disorders, such as ulcerative colitis (UC) and Crohn’s disease (CD). Additionally, the disruption of gut microbiota has been implicated in certain cancers. Thus, there exists a need for more effective methods for restoring disrupted gut bacteria flora in the treatment of a variety of diseases and conditions, including diseases and conditions of the digestive tract and cancer.Atty. Docket No. 052209-0664SUMMARY

[0008] Provided herein in accordance with some aspects are methods for administering a microbiota restoration composition by colonoscopy. These methods are useful for treating diseases and / or conditions of the digestive tract, such as Clostridioides difficile infection (CDI), including recurrent CDI, for treating other gastrointestinal diseases or disorders, such as ulcerative colitis (UC) and Crohn’s disease (CD), and for treating cancer, and to achieve other beneficial results as disclosed herein.

[0009] In accordance with some aspects, provided are methods for treating recurrent Clostridioides difficile infection (CDI) in a human patient in need thereof, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline. Prior to treatment with the microbiota restoration therapy composition the patient may have had one or more episodes of a recurrent CDI. Prior to treatment with the microbiota restoration therapy composition, the patient may have been identified as having had one or more episodes of a recurrent CDI. Prior to treatment with the microbiota restoration therapy composition, the patient may have been identified as having had a stool test positive for C. difficile or C. difficile toxin associated with the CDI. Prior to treatment with the microbiota restoration therapy composition, the patient may have completed a course of antibiotic therapy for treatment of CDI and optionally may have been identified as having completed at least one course of antibiotic therapy for treatment of CDI, in either case where the course of antibiotic therapy may have comprised at least 10 days of antibiotic therapy, and where, additionally or alternatively, the microbiota restoration therapy composition may be administered 24 to 72 hours after a last dose of the antibiotic therapy for CDI.

[0010] In accordance with some aspects, provided are methods for treating a gastrointestinal disease or disorder in a human patient in need thereof, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol andAtty. Docket No. 052209-0664 saline. The gastrointestinal disease or disorder may be one or more selected from ulcerative colitis and Crohn’s disease, optionally wherein the gastrointestinal disease or disorder is mild to moderate ulcerative colitis. The gastrointestinal disease or disorder may be one or more selected from irritable bowel syndrome, spastic colon, functional bowel disease (FBD), non-ulcer dyspepsia (NUD), gastroesophageal reflux, inflammatory bowel disease, idiopathic or simple constipation, diverticular disease, AIDS enteropathy, small bowel bacterial overgrowth, celiac / coeliac disease, polyposis coil, colonic polyps, chronic idiopathic pseudo obstructive syndrome, chronic gut infections with specific pathogens, and viral gastrointestinal disorders.

[0011] In accordance with some aspects, provided are methods for treating a human patient suffering from cancer, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

[0012] In accordance with some aspects, provided are methods for decolonizing patients that carry antibiotic resistance genes (ARGs), e.g. , in their gut microbiota, e.g., to achieve antimicrobial resistance (AMR) gut decolonization, e.g., to reduce antimicrobial resistance gene abundance and / or reduce the presence of antimicrobial resistant organisms, wherein the methods comprise administering a dose of a microbiota restoration therapy composition to the patient rectally or by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, optionally formulated in a solution of polyethylene glycol and saline.

[0013] In accordance with some aspects, provided herein are methods for altering the gut microbiota composition in a human patient in need thereof, wherein the methods comprise administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, optionally formulated in a solution of polyethylene glycol and saline.

[0014] In accordance with some aspects, provided herein are methods for altering the gut metabolome composition in a human patient in need thereof, wherein the methods compriseAtty. Docket No. 052209-0664 administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, optionally formulated in a solution of polyethylene glycol and saline.

[0015] In accordance with any of the foregoing aspects, the dose of the composition may comprise from about I O8to about 5 * IO10colony-forming units (CFU) / mL of fecal microbes.

[0016] In accordance with any of the foregoing aspects, the fecal microbes may comprise sporeforming microbes and non-spore forming microbes, such as microbes of species Bacteroides. In accordance with any of the foregoing aspects, the dose of the composition may comprise > IxlO5CFU / mL of Bacteroides.

[0017] In accordance with any of the foregoing aspects, the fecal microbes may be obtained by a process comprising processing a donor human fecal sample with a solution comprising polyethylene glycol and saline, and obtaining a fdtrate comprising the fecal microbes.

[0018] In accordance with any of the foregoing aspects, the solution may comprise from 20 g to 50 g polyethylene glycol per 1 L of saline, or 30 g polyethylene glycol per 1 L of saline.

[0019] In accordance with any of the foregoing aspects, the polyethylene glycol may comprise polyethylene glycol 3350.

[0020] In accordance with any of the foregoing aspects, the saline may be a 0.9% solution of sodium chloride.

[0021] In accordance with any of the foregoing aspects, the dose of the composition may have a volume of about 150 mb.

[0022] In accordance with any of the foregoing aspects, the dose of the composition may comprise no more than about 6 grams of polyethylene glycol.

[0023] In accordance with any of the foregoing aspects, the dose of the composition comprises no more than 5.97 grams of polyethylene glycol in saline.Atty. Docket No. 052209-0664

[0024] In accordance with any of the foregoing aspects, the dose of the composition may comprise from about 150 g to about 192 g of the solution of polyethylene glycol in saline.

[0025] In accordance with any of the foregoing aspects, the dose of the composition, the dose of the composition may have a microbial colony forming unit (CFU) count of at least about 10 CFU per plate at a serial dilution of 104when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate.

[0026] In accordance with any of the foregoing aspects, the dose of the composition may have a microbial colony forming unit (CFU) count of from about 2.0 x 107CFU per mL to about 5.0 x 1010CFU per mL when cultured on a Center for Disease Control (CDC) culture plate.

[0027] In accordance with any of the foregoing aspects, the composition may have bacteria from at least three colony types when cultured on a Center for Disease Control (CDC) culture plate.

[0028] In accordance with any of the foregoing aspects, the composition may include bacteria from at least seven different families.

[0029] In accordance with any of the foregoing aspects, the dose of the composition may have a Shannon Diversity Index of 0.4-2.5 when calculated at the family level.

[0030] In accordance with any of the foregoing aspects, the dose of the composition the composition may be administered via colonoscopy to the right side of the colon, including between the ileocecal valve and the hepatic flexure of the right side of the colon.

[0031] In accordance with any of the foregoing aspects, prior to treatment with the microbiota restoration therapy composition, the patient may have been identified as not having experienced CDI diarrhea for two consecutive days.

[0032] In accordance with any of the foregoing aspects, the patient does not experience CDI diarrhea for at least 8 weeks following treatment with the microbiota restoration therapy composition.

[0033] In accordance with any of the foregoing aspects, efficacy of methods as described herein comprising administration of a microbiota restoration therapy composition by colonoscopy is atAtty. Docket No. 052209-0664 least non-inferior to a method comprising administering the microbiota restoration therapy composition by enema. In some embodiments, methods of treating CDI as described herein comprising administration of a microbiota restoration therapy composition by colonoscopy have a clinical efficacy rate of 75% of greater, such as a clinical efficacy rate of about 75-100%, including a clinical efficacy rate of 95% of greater or a clinical efficacy rate of 100%.

[0034] In accordance with any of the foregoing aspects, the treatment may reduce ARG richness, including reducing one or more clinically important ARG groups selected from gram-negative beta-lactam resistance-associated ARG groups, fluoroquinolone resistance-associated ARG groups, colistin resistance-associated ARG groups, and vancomycin-resistance-associated ARG groups, such as one or more selected from beta-lactam ARG richness, fluoroquinolone ARG richness, glycopeptide ARG richness, and colistin ARG richness. Additionally or alternatively, the treatment may reduce one or more clinically critical ARG groups including one or more selected from clinically critical gram-negative beta-lactam resistance ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF) and clinically critical colistin resistance ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ). In some aspects the treatment may increase tetracycline ARG richness.

[0035] In accordance with any of the foregoing aspects, the patient may be immunocompromised, such as by having a disease or condition rendering them immunocompromised and / or by being treated with an immunocompromising medication. In some embodiments, an immunocompromised patient has one or more diseases or conditions selected from neoplasms, renal and urinary disorders, immune system disorders, HIV infection, and thalassemia at the time of treatment. In some embodiments, an immunocompromised patient has been treated with one or more immunocompromising medications for 2 weeks or longer before treatment with the microbiota restoration therapy composition and optionally continues immunosuppressive treatment throughout treatment with the microbiota restoration therapy composition and further optionally thereafter.

[0036] In accordance with any of the foregoing aspects, the treatment may result in improvement in quality of life relating to one or more of physical, mental and social domains of the C. diff Health-Related Quality -of-Life (Cdiff32) questionnaire. In some aspects, the treatment results inAtty. Docket No. 052209-0664 improvement in quality of life relating to one or more of anxiety, coping with disease / health perception, control of disease, impact of stress, dysphoria, relationships, social reaction, daily activities, diet, sleep, and discomfort.

[0037] In accordance with any of the foregoing aspects, the treatment may increase the relative abundance of one or more of Clostridia and Bacteroidia in the subject’s gut microbiome. In some aspects, the treatment increases the relative abundance of one or more of Blautia, Lachnosclostridium, Faecali bacterium, Roseburia, Eubacterium, and Bacteroides in the subject’s gut microbiome.

[0038] In accordance with any of the foregoing aspects, the treatment may decrease the relative abundance of one or more of Gammaproteobacteria, Bacilli and Negativi cutes in the subject’s gut microbiome. In some aspects, the treatment decreases the relative abundance of one or more of Klebsiella, Escherichia, Citrobacter, Lactobacillus, and Streptococcus in the subject’s gut microbiome.

[0039] In accordance with any of the foregoing aspects, the treatment may increase the ratio of secondary bile acids to primary bile acids (S:P) in the subject’s gut metabolome. In some aspects, the method increases one or more of deoxycholic acid (DCA) and lithocholic acid (LCA) in the subject’s gut metabolome. In some aspects the method decreases one or more of cholic acid (CA), glycocholic acid (GA), and taurocholic acid (TCA) in the subject’s gut metabolome.

[0040] Thus, provided in one aspect is a method for treating recurrent C. difficile infection (CDI) in a human patient in need thereof, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

[0041] Provided in another aspect are methods for altering the gut microbiota composition in a human patient in need thereof, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, optionally formulated in a solution of polyethylene glycol and saline.Atty. Docket No. 052209-0664

[0042] Provided in another aspect are methods for altering the gut metabolome composition in a human patient in need thereof, wherein the methods comprise administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, optionally formulated in a solution of polyethylene glycol and saline.

[0043] Provided in another aspect are methods for decolonizing patients that carry antibiotic resistance genes (ARGs), e.g., in their gut microbiota, e.g., to achieve antimicrobial resistance (AMR) gut decolonization, e.g., to reduce antimicrobial resistance gene abundance and / or reduce the presence of antimicrobial resistant organisms, wherein the methods comprise administering a dose of a microbiota restoration therapy composition to the patient rectally or by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, optionally formulated in a solution of polyethylene glycol and saline.

[0044] Provided in another aspect are methods for treating a gastrointestinal disease or disorder in a human patient in need thereof, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

[0045] Provided in another aspect are methods for treating a human patient suffering from cancer, comprising administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

[0046] In some embodiments, the dose of the composition comprises from about 1 x 108to about 5* 1010colony-forming units (CFU) / mL of fecal microbes. In some embodiments, the fecal microbes comprise spore-forming microbes and non-spore forming microbes. In some embodiments, the fecal microbes comprises microbes of species Bacteroides. In some embodiments, the dose of the composition comprises > IxlO5CFU / mL of Bacteroides.Atty. Docket No. 052209-0664

[0047] In some embodiments, the fecal microbes are obtained by a process comprising processing a donor human fecal sample with a solution comprising polyethylene glycol and saline, and obtaining a filtrate comprising the fecal microbes. In some embodiments, the solution comprises from 20 g to 50 g polyethylene glycol per 1 L of saline. In some embodiments, the solution comprises 30 g polyethylene glycol per 1 L of saline. In some embodiments, the polyethylene glycol comprises polyethylene glycol 3350. In some embodiments, the saline is a 0.9% solution of sodium chloride. In some embodiments, the dose of the composition has a volume of about 150 mL. In some embodiments, the dose of the composition comprises no more than about 6 grams of polyethylene glycol. In some embodiments, dose of the composition comprises no more than 5.97 grams of polyethylene glycol in saline. In some embodiments, the dose of the composition comprises from about 150 g to about 192 g of the solution of polyethylene glycol in saline.

[0048] In some embodiments, the dose of the composition has a microbial colony forming unit (CFU) count of at least about 10 CFU per plate at a serial dilution of 104when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate. In some embodiments, the dose of the composition has a microbial colony forming unit (CFU) count of from about 2.0 x 107CFU per mL to about 5.0 x 1010CFU per mL when cultured on a Center for Disease Control (CDC) culture plate.

[0049] In some embodiments, the dose of the composition has bacteria from at least three colony types when cultured on a Center for Disease Control (CDC) culture plate. In some embodiments, the composition includes bacteria from at least seven different families. In some embodiments, the composition has a Shannon Diversity Index of 0.4-2.5 when calculated at the family level.

[0050] In some embodiments, prior to treatment with the microbiota restoration therapy composition the patient has had one or more episodes of a recurrent CDI. In some embodiments, prior to treatment with the microbiota restoration therapy composition the patient has been identified as having had one or more episodes of a recurrent CDI. In some embodiments, prior to treatment with the microbiota restoration therapy composition, the patient has been identified as having had a stool test positive for C. difficile or C. difficile toxin associated with the CDI.Atty. Docket No. 052209-0664

[0051] In some embodiments, the patient has completed a course of antibiotic therapy for treatment of CDI prior to treatment with the microbiota restoration therapy composition. In some embodiments, the patient has been identified as having completed at least one course of antibiotic therapy for treatment of CDI prior to treatment with the microbiota restoration therapy composition. In some embodiments, the course of antibiotic therapy comprised at least 10 days of antibiotic therapy. In some embodiments, the microbiota restoration therapy composition is administered 24 to 72 hours after a last dose of the antibiotic therapy for CDI.

[0052] In some embodiments, prior to treatment with the microbiota restoration therapy composition the patient has been identified as not having experienced CDI diarrhea for two consecutive days.

[0053] In some embodiments, the patient is immunocompromised, such as by having a disease or condition rendering them immunocompromised and / or by being treated with an immunocompromising medication.

[0054] In some embodiments, the composition is administered via colonoscopy to the right side of the colon. In some embodiments, the composition is administered via colonoscopy between the ileocecal valve and the hepatic flexure of the right side of the colon.

[0055] In some embodiments, the patient does not experience CDI diarrhea for at least 8 weeks following treatment with the microbiota restoration therapy composition. In some embodiments, efficacy of a method as described herein comprising administration of a microbiota restoration therapy composition by colonoscopy is at least non-inferior to a method comprising administering the microbiota restoration therapy composition by enema.

[0056] In some embodiments, the treatment reduces ARG richness, including reducing one or more clinically important ARG groups selected from gram-negative beta-lactam resistance- associated ARG groups, fluoroquinolone resistance-associated ARG groups, colistin resistance- associated ARG groups, and vancomycin-resistance-associated ARG groups, such as one or more selected from beta-lactam ARG richness, fluoroquinolone ARG richness, glycopeptide ARG richness, and colistin ARG richness. Additionally or alternatively, the treatment may reduce one or more clinically critical ARG groups including one or more selected from clinically critical gram-Atty. Docket No. 052209-0664 negative beta-lactam resistance ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF) and clinically critical colistin resistance ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ). In some aspects the treatment increases tetracycline ARG richness.

[0057] In any of the foregoing embodiments, treatment with a microbiota restoration therapy composition as described herein may result in improvement in the quality of life of the patient, such as improvement in quality of life relating to one or more of the physical, mental and social domains of the C. diff Health-Related Quality-of-Life (Cdiff32) questionnaire. In some aspects, treatment as described herein results in improvement in quality of life relating to one or more of anxiety, coping with disease / health perception, control of disease, impact of stress, dysphoria, relationships, social reaction, daily activities, diet, sleep, and discomfort.

[0058] In any of the foregoing embodiments, treatment with a microbiota restoration therapy composition as described herein may increase the relative abundance of bacteria of one or more of Clostridia and Bacteroidia, e.g., in the subject’s gut microbiome. In some aspects, treatment increases the relative abundance of one or more of Blautia, Lachnosclostridium, Faecalibacterium , Roseburia, Eubacterium, and Bacteroides.

[0059] In any of the foregoing embodiments, treatment with a microbiota restoration therapy composition as described herein may decrease the relative abundance of one or more of Gammaproteobacteria, Bacilli and Negativicutes, e.g., in the subject’s gut microbiome. In some aspects, treatment decreases the relative abundance of one or more of Klebsiella, Escherichia, Citrobacter, Lactobacillus, and Streptococcus.

[0060] In any of the foregoing embodiments, treatment with a microbiota restoration therapy composition as described herein may increase the ratio of secondary bile acids to primary bile acids (S:P) e.g., in the subject’s gut metabolome. In some aspects, the treatment increases the concentration of one or more of deoxycholic acid (DCA) and lithocholic acid (LCA). In some aspects the method decreases the concentration one or more of cholic acid (CA), glycocholic acid (GA), and taurocholic acid (TCA).Atty. Docket No. 052209-0664

[0061] The above summary of some embodiments is not intended to describe each disclosed embodiment or every implementation of the present disclosure, which is more fully described by the following detailed description and illustrated by the drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 illustrates an example sample bag.

[0063] FIG. 2 illustrates an example tube assembly.

[0064] FIG. 3 illustrates the trial design diagram of the study of Example 2.

[0065] FIG. 4 illustrates the trial design of the study of Example 7.

[0066] FIG. 5 illustrates gut microbiome composition for treatment responders and placebo responders from baseline to 6 months after treatment of the study of Example 7.

[0067] FIG. 6A illustrates multiple ARG classes detected at baseline for treatment responders, and placebo responders of the study of Example 7, as compared to healthy subjects.

[0068] FIG. 6B illustrates clinically important ARG classes detected at baseline, which included beta-lactam ARGs (Beta.), tetracycline ARGs (Tetra.), glycopeptide ARGs (Glyco.), and fluoroquinolone ARGs (Fluro.).

[0069] FIG. 7 shows ARG class richness reduced after treatment in the study of Example 7.

[0070] FIG. 8 illustrates richness changes for clinically important ARG classes including betalactam ARGs, tetracycline ARGs, glycopeptide ARGs, and fluoroquinolone ARGs, after treatment in the study of Example 7.

[0071] FIGs. 9 A and 9B illustrate ARG richness for ARGs grouped at mechanism and group levels respectively, which decreased significantly after treatment in the study of Example 7.

[0072] FIG. 10 illustrates reductions in clinically critical ARG groups after treatment in the study of Example 7.Atty. Docket No. 052209-0664

[0073] FIG. 11 illustrates the C. difficile health-related quality of life survey (Cdiff32 HRQOL Questionnaire) described in Example 8. The survey is adapted from Garey et al., J. Clin. Gastroenterol. 2016; 50:631-637.

[0074] FIG. 12 illustrates Cdiff32 questionnaire response patterns for changes in categorical responses from baseline to Week 8 in the study of Example 8.

[0075] FIG. 13 illustrates the count of Cdiff32 questionnaire responses for each question at the first timepoint available for each participant in the study of Example 8. Larger circles represent higher proportions of categorical responses from highest (HRQOL A) to the lowest HRQOL (HRQOL E).

[0076] FIG. 14 illustrates summaries of repeated measures conditional logistic regression analysis comparing the probability of improved response after treatment with a microbiota restoration therapy composition (RBL) versus placebo administration in Example 8, with an odds ratio greater than 1 indicative that responses were more likely to have improved in the RBL treatment group. Domain summaries account for repeated measures.

[0077] FIG. 15 illustrates summaries of Cdiff32 scores of treatment non-responders from the study in Example 8. Domain summaries account for repeated measures.

[0078] FIG. 16 illustrates microbiome partitioning analysis of class-level taxonomic composition based on Cdiff32 questionnaire mental domain HRQOL scoring categories for participants from the study in Example 8. Panel A shows the optimal DM-RPart tree generated by fitting and cross-validation pruning. Responses to Cdiff32 questions defined splitting rules and were standardized as per FIG. 12. Terminal nodes are ordered from higher HRQOL scores (left side of tree) to lower HRQOL scores (right side of tree) as per FIG. 12. Numbers in parentheses reflect the Cidff32 questionnaire number. Panel B shows a barchart comparing taxonomic composition for each of the 8 terminal nodes. Panel C shows the distribution of Cdiff32 HRQOL mental domain scores for all questions for each of the 8 terminal nodes. Larger circles represent higher proportions of categorical responses from highest (HRQOL A) to the lowest HRQOL (HRQOL E).Atty. Docket No. 052209-0664

[0079] FIG. 17 illustrates the genera that drive class-level differences among terminal nodes from the microbiome partitioning analysis in FIG. 16, Panels A B, stratified by the four indicated Microbiome Health Index (MHI) classes.

[0080] FIG. 18 illustrates the Dirichlet-multinomial recursive partitioning model (DM-RPart) analysis of bile acid (BA) composition and Cdiff32 HRQOL mental domain categories for participants from the study in Example 8. Panel A shows the optimal DM-RPart tree generated by fitting and cross-validation pruning. Responses to Cdiff32 questions defined splitting rules and were standardized as per FIG. 12. Terminal nodes are ordered from higher HRQOL scores (left side of tree) to lower HRQOL scores (right side of tree) as per FIG. 12. Numbers in parentheses reflect the Cidff32 questionnaire number. Panel B shows the distribution of secondary to primary BA ratio (S :P ratio) for each of the four terminal nodes plotted on a loglO y-axis. Panel C shows the Forest plot comparing relative abundance of four BAs associated with colonization resistance (CA, DCA, GCA, LCA, and TCA) for each of the four terminal nodes. CA, cholic acid; DCA, deoxycholic acid; GCA, glycholic acid; LCA, lithocholic acid; TCA, taurocholic acid.

[0081] FIG. 19 illustrates the distribution of Cdiff32 HRQOL mental domain scores for all questions for each of the 4 terminal nodes generated from the Dirichlet-multinomial recursive partitioning model (DM-RPart) analysis of BA composition (secondary to primary BA ratio; S:P ratio) in FIGs. 18, Panels A and B. Larger circles represent higher proportions of categorical responses from highest to the lowest HRQOL.DETAILED DESCRIPTION

[0082] This disclosure relates in some aspects to methods for establishing or re-establishing a healthy microbial population of gastrointestinal microbiota by administering fecal microbes obtained from a healthy donor by colonoscopy, or rectally (e.g., by enema). The methods disclosed herein comprise administering a composition comprising fecal microbes from a human fecal sample obtained from a healthy donor (e.g., a microbiota restoration therapy composition). Using a microbiota restoration therapy composition to restore healthy gut microbiota may be used to treat diseases and / or conditions of the digestive tract, such as CDI, other gastrointestinal diseases or disorders, such as UC and CD, and other conditions such as cancer. Also disclosed are methods for altering the gut microbiota composition in a human patient in need thereof using a microbiotaAtty. Docket No. 052209-0664 restoration therapy composition as described herein. Also disclosed are methods for altering the gut metabolome in a human patient in need thereof using a microbiota restoration therapy composition as described herein. Also disclosed are methods and uses of microbiota restoration therapy to decolonize patients that carry antibiotic resistance genes (ARGs), e.g., in their gut microbiota to achieve antimicrobial resistance (AMR) gut decolonization, e.g., to reduce antimicrobial resistance gene abundance and / or reduce the presence of antimicrobial resistant organisms, comprising administering a microbiota restoration therapy composition rectally or by colonoscopy. The methods may be effective for improving quality of life in treated patients, such as may be assessed by methodology described herein. The methods may alter the gut microbiota composition and / or the gut metabolome composition.

[0083] The present disclosure provides methods for treating diseases and / or conditions of the digestive tract, such as C. difficile infection (CDI), including recurrent CDI, by administering a dose of a microbiota restoration therapy composition by colonoscopy. As reported in Example 2 below, such methods (e.g., comprising administering a microbiota restoration therapy composition as described herein by colonoscopy) have a surprisingly and unexpectedly high clinical efficacy rate in the treatment of CDI, even as compared to treatment by administering the microbiota restoration therapy rectally. For example, as reported in Example 2, the interim analysis of a clinical trial of a microbiota restoration therapy composition as described herein administered by colonoscopy showed 100% clinical efficacy in treating CDI. In contrast, a clinical trial of a microbiota restoration therapy composition administered by enema was reported to have a clinical efficacy of 70.6% in treating CDI. See, Khanna, et al., Drugs 2022; 82: 1527-1538.

[0084] Microbiota restoration therapy (MRT) (administration of human fecal microbiota from healthy donors) is believed to restore the diversity of the gut microbiome, and for CDI, thereby suppress C. difficile outgrowth, treat CDI, and reduce the risk of recurrence of CDI. In some aspects, the treatment may alter the gut microbiota composition and / or the gut metabolome composition, as described in more detail below. In some aspects, the treatment may reduce ARG richness, including reducing one or more clinically important ARG groups selected from gramnegative beta-lactam resistance-associated ARG groups, fluoroquinolone resistance-associated ARG groups, colistin resistance-associated ARG groups, and vancomycin-resistance-associated ARG groups, such as one or more selected from beta-lactam ARG richness, fluoroquinolone ARGAtty. Docket No. 052209-0664 richness, glycopeptide ARG richness, and colistin ARG richness. Additionally or alternatively, the treatment may reduce one or more clinically critical ARG groups including one or more selected from clinically critical gram-negative beta-lactam resistance ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF) and clinically critical colistin resistance ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ). In some aspects the treatment may increase tetracycline ARG richness.

[0085] In some aspects, also provided are methods for treating other gastrointestinal diseases or disorders, such as ulcerative colitis (UC) and Crohn’s disease (CD) by administering a dose of a microbiota restoration therapy composition by colonoscopy or rectally. In other aspects, also provided are methods for treating cancer by administering a dose of a microbiota restoration therapy composition by colonoscopy or rectally.

[0086] In some instances, administration of a microbiota restoration therapy composition by colonoscopy as described herein may be preferred or advantageous on account of one or more of the following factors: improved convenience and / or patient comfort over other routes of administration, such as by enema; the administering physician’s familiarity with the colonoscopy route of administration; the medical practice’s familiarity with and support for colonoscopy procedures; guidelines in favor of administration of fecal matter transplants and microbiome restoration therapies by colonoscopy; clinical efficacy.1. Definitions

[0087] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present invention pertains, unless otherwise defined. Any suitable materials and / or methods known to those of ordinary skill in the art can be utilized in carrying out the present invention in view of the guidance provided herein; however, specific materials and methods are described for illustrative purposes. Materials, reagents and the like to which reference is made in the following description and examples are obtainable from commercial sources, unless otherwise noted.Atty. Docket No. 052209-0664

[0088] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only. As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0089] As used herein, “about” when used with a numerical value means the numerical value stated as well as plus or minus 10% of the numerical value. For example, “about 10” should be understood as both “10” and “9-11.”

[0090] The recitation of numerical ranges by endpoints includes all value within that range (e.g. “1 to 5” includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.).

[0091] As used herein, a phrase in the form “A / B” or in the form “A and / or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0092] As used herein, the terms “comprising,” “including,” and “containing” are used expansively to mean that the described compositions, methods, or kits include at least the stated elements, and may include other elements that are not specified.

[0093] As used herein, “subject” and “patient” are used interchangeably and denote any mammal, including humans. For example, a subject may be suffering from or at risk of developing a condition that can be treated or prevented with a microbiota restoration therapy composition.

[0094] The terms “administer,” “administration,” and “administering” as used herein refer to providing, giving, dosing and / or prescribing, such as by a health professional or his or her authorized agent or under his or her direction, and putting into, taking, or consuming, such as by a health professional or the subject.

[0095] The terms “treat,” “treating,” and “treatment” as used herein include alleviating, abating or ameliorating a disease or condition, reducing the risks thereof, or reducing the risk of recurrence thereof, whether or not the disease or condition is considered to be “cured” or “healed,” and whether or not all symptoms are resolved.Atty. Docket No. 052209-0664

[0096] As used herein, the phrases “therapeutically effective amount” and “therapeutically effective dose” refer to an amount or dose that provides the specific pharmacological effect for which the drug is administered in a subject in need of such treatment. It is emphasized that a therapeutically effective amount will not always be effective in treating the targeted condition in a given patient, even though such amount or dose is deemed to be a therapeutically effective amount or dose by those of skill in the art. For convenience only, exemplary doses and therapeutically effective amounts are provided below with reference to adult human subjects. Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject and / or condition / disease.

[0097] When particular features, structures, and / or characteristics are described in connection with one embodiment, it should be understood that such features, structures, and / or characteristics may also be present in or used connection with other embodiments whether or not explicitly described unless clearly stated to the contrary.2. Methods of Treatment

[0098] As noted above, the present disclosure provides methods comprising administering a dose of a microbiota restoration therapy (MRT composition) by colonoscopy or rectally (e.g., by enema). As described in more detail below, the MRT composition comprises fecal microbes obtained from a human fecal sample from a healthy donor. In specific embodiments, the composition is administered by colonoscopy to the right side of the colon (such as between the ileocecal valve and the hepatic flexure of the colon). Thus, in some embodiments, the composition is administered by colonoscopy to the right side of the colon. In some embodiments, the composition is administered by colonoscopy between the ileocecal valve and the hepatic flexure of the right side of the colon. In other embodiments, the composition is administered rectally (e.g., by enema).

[0099] In some embodiments, subjects treated by the methods described herein have completed at least one course of antibiotic therapy prior to MRT treatment, such as being subjects identified as having completed at least one course of antibiotic therapy prior to MRT. Antibiotic pretreatment may decrease colonization resistance after administration of a MRT composition as described herein.Atty. Docket No. 052209-0664

[0100] For example, prior antibiotic therapy may comprise a course of antibiotic treatment. For example, the course of antibiotic treatment may comprise administering an antibiotic, e g., vancomycin, daily for 6 days, such as at a dose of, or equivalent to, 4* 1 oral capsule (4* 125 mg) per day. In some embodiments, subjects treated with antibiotic therapy have an antibiotic washout period of at least 24 hours but no more than 72 hours prior to administration of a MRT composition as described herein. Thus, in some embodiments, prior to treatment with a MRT composition as described herein, the subject has had a course of antibiotic therapy, optionally comprising at least 6 days of antibiotic therapy. In further specific embodiments, the MRT composition is administered 24 to 72 hours after a last dose of the antibiotic therapy.

[0101] The methods described herein also may be particularly suited for patients who immunocompromised, such as by having a disease or condition rendering them immunocompromised and / or by being treated with an immunocompromising medication. Immunocompromised patients include those having one of more medical conditions associated with impaired immune function, such as end-stage renal disease, renal failure, asplenia, human immunodeficiency virus (HIV) or HIV infection, congenital hemoglobinopathies, immunodeficiency syndromes, and end-stage liver disease. Specific examples of immunocompromising conditions include but are not limited to neoplasms (such as malignant and unspecified), renal and urinary disorders (such as end stage renal disease and renal failure), immune system disorders, HIV infection, and thalassemia.

[0102] Immunocompromised patients also include patients being treated with one or more immunocompromising medications, such as patients treated with one or more immunocompromising medications for 2 weeks or longer before treatment with a microbiota restoration therapy composition as described herein who optionally continue immunosuppressive treatment throughout treatment with the microbiota restoration therapy composition and further optionally thereafter. Examples of immunocompromising medications include glucocorticoids, tumor necrosis factor alpha inhibitors, calcineurin inhibitors, interleukin inhibitors, purine analogues, monoclonal antibodies, protein kinase inhibitors, anthracy clines and related substances, interferons, antineoplastic agents, platinum compounds, pyrimidine analogues, colony stimulating factors, combination of antineoplastic agents, nitrogen mustard analogues, podophyllotoxin derivatives, and vinca alkaloids and analogues. Specific examples of immunocompromisingAtty. Docket No. 052209-0664 glucocorticoids include prednisone, cortisone, dexamethasone, hydrocortisone, methylprednisolone, methylprednisolone sodium succinate, prednisolone, prednisone, and triamcinolone. As an example, a dose of > 20 mg prednisone / day or equivalent amount of another immunocompromising medication (see Table 10 in Example 9 below) may be considered to be an immunocompromising dose as used herein. Patients taking immunocompromising medications may include those having one or more immunocompromising diseases or conditions. Additionally or alternatively, patients taking immunocompromising medications may include those having one or more diseases or conditions selected from inflammatory bowel disease, arthritis, solid organ transplantation, cancer, systemic lupus erythematosus, multiple sclerosis, psoriasis, and scleroderma.3. Treatment of CDI

[0103] Provided in accordance with one aspect of the present disclosure are methods for treating diseases and / or conditions of the digestive tract, such as for treating C. difficile infection (CDI), including recurrent CDI.

[0104] Conventional treatments for CDI utilize antibiotics, which further disrupt the gut bacteria flora and limit the recovery of the gut microbiota following CDI. This decreased diversity of the gut microbiota following antibiotic treatment can predispose patients to re-colonization with C. difficile and lead to recurrent infections.

[0105] Recurrent CDI (rCDI) is defined clinically as recurrence of symptoms within 8 weeks after response to, and cessation of, specific antibiotic treatment, with a positive diagnostic test for C. difficile and exclusion of other enteropathogens. Recurrent CDI (rCDI) presents most commonly as repeated bouts of diarrhea that respond to antibiotic treatment but recur within days to 8 weeks of stopping the antibiotic treatment. As noted above, repeated bouts of infection can continue for years, leading to persistent use of antibiotics, markedly reduced quality of life (QoL), repeated hospitalizations, and even death.

[0106] It is estimated that the risk of a first recurrence after an initial response to antibiotic therapy for a primary CDI ranges from 20 to 30%. In patients with prior rCDI, the risk of anAtty. Docket No. 052209-0664 additional recurrence increases to 40 to 60%. Analyses of recurrence history over time suggest that the rate of recurrence for any number of recurrences after the first recurrence is similar.

[0107] Immunocompromised individuals are at increased risk for primary CDI and recurrence due to factors such as frequent interactions with health care systems and increased exposure to antibiotics, in addition to additional risk conferred by being immunocompromised. In particular, a prospective cohort study found that immunocompromising conditions double the risk of rCDI 90 days after the primary episode. See, e.g., Avni, et al., Int. J. Infect. Dis. 2020; 90: 237-242. Thus, in some embodiments, a subjected being treated for CDI in accordance with the present disclosure is immunocompromised, such as by having a disease or condition rendering them immunocompromised and / or by being treated with an immunocompromising medication.

[0108] As noted above, in accordance with some aspects, the methods disclosed herein comprise administering a dose of a microbiota restoration therapy (MRT) composition by colonoscopy. Alternatively, the methods disclosed herein may comprise administering a dose of a microbiota restoration therapy (MRT) composition rectally (e.g., by enema). Without being bound by theory, it is believed that the administered fecal microbes repopulate the gut microbiota, restore diversity of the gut microbiome, and suppress C. difficile outgrowth, thereby treating CDI and reducing the risk of recurrence of CDI.

[0109] The methods described herein are particularly suited for patients having recurrent CDI. In some instances, recurrent CDI prior to treatment is defined as the documented diagnosis of(1) CDI diarrhea, i.e., the passage of >3 unformed / loose stools in 24 consecutive hours for >2 consecutive days, that began within 8 weeks after completion of previous CDI treatment and(2) >1 stool test positive for toxigenic C. difficile or C. difficile toxin, documented at the time of the CDI diarrhea for the qualifying rCDI episode. In some instances, recurrent CDI during treatment is defined as (1) passage of >3 unformed / loose stools (i.e., Bristol Stool Scale type 6-7) in 24 consecutive hours for >2 consecutive days and (2) a stool test collected before start of any antibiotic treatment being positive for C. difficile toxin, as determined by a toxin protein detecting assay at the time of the diarrhea.

[0110] Thus, patients who may be treated by the methods described herein include, but are not limited to, those having one or more or all of the following prior to treatment with the microbiotaAtty. Docket No. 052209-0664 restoration therapy composition: have experienced one or more episodes of a recurrent CDI, have been identified as having had one or more episodes of a recurrent CDI, have been identified as having had a stool test positive for C. difficile or C. difficile toxin associated with the CDI, have completed a course of antibiotic therapy for treatment of CDI, have been identified as having completed at least one course of antibiotic therapy for treatment of CDI, have been identified as not having experienced CDI diarrhea for two consecutive days, are immunocompromised and / or have been identified as being immunocompromised.[0U1] In some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has had one or more episodes of a recurrent CDI. Additionally, or alternatively, in some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has been identified as having had one or more episodes of a recurrent CDI. Additionally, or alternatively, in some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has been identified as having had a stool test positive for C. difficile or C. difficile toxin associated with the CDI. Additionally, or alternatively, in some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has completed a course of antibiotic therapy for treatment of CDI. Additionally, or alternatively, in some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has been identified as having completed at least one course of antibiotic therapy for treatment of CDI. Additionally, or alternatively, in some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has been identified as not having experienced CDI diarrhea for two consecutive days.

[0112] With regard to antibiotic treatment prior to administration of a microbiota restoration therapy composition as described herein, patients may have completed antibiotic treatment for CDI comprising at least 10 consecutive days within the last 30 days. Preferably, patients are subject to an antibiotic washout period of at least 24 hours but no more than 72 hours prior to administration of a microbiota restoration therapy composition as described herein. Thus, in some embodiments, prior to treatment with a microbiota restoration therapy composition as described herein, the patient has had a course of antibiotic therapy for CDI comprising at least 10 days of antibioticAtty. Docket No. 052209-0664 therapy. In further specific embodiments, the microbiota restoration therapy composition is administered 24 to 72 hours after a last dose of the antibiotic therapy for CDI.

[0113] For the methods described herein for treating CDI, treatment success may be assessed with reference to the absence of CDI diarrhea (passage of > 3 unformed / loose stools [i.e., Bristol Stool Scale type 6-7] in 24 consecutive hours for > 2 consecutive days and a stool test collected before start of any antibiotic treatment, which is positive for C. difficile toxin, as determined by a toxin protein detecting assay at the time of the diarrhea) for a period of time, such as for 8 weeks after treatment. Thus, in some embodiments, the patient does not experience CDI diarrhea for at least 8 weeks following treatment with a microbiota restoration therapy composition as described herein.

[0114] In some embodiments, efficacy of a method as described herein comprising administration of a microbiota restoration therapy composition by colonoscopy is at least noninferior to a method comprising administering the microbiota restoration therapy composition by enema.

[0115] In some embodiments, the methods described herein for treating CDI comprising administration of a microbiota restoration therapy composition as described herein by colonoscopy have a clinical efficacy rate of at least 75%, including a clinical efficacy rate of 75% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100%, as may be determined by the absence of CDI diarrhea for 8 weeks after treatment. For example, the methods described herein for treating CDI may have a clinical efficacy rate of about 75-100%, about 80-100%, about 90-100%, or about 95-100%. Thus, in some embodiments, at least 75% of subjects treated for CDI by a method as described herein comprising administration of a microbiota restoration therapy composition as described herein by colonoscopy achieve treatment success, as may be determined by the absence of CDI diarrhea for 8 weeks after treatment, including 75% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% of subjects achieving treatment success, such as about 75-100%, about 80-100%, about 90-100%, or about 95-100% of subjects achieving treatment success, as may be determined by the absence of CDI diarrhea for 8 weeks after treatment. As noted above, a clinical trial of a microbiota restoration therapy composition administered by enemaAtty. Docket No. 052209-0664 was reported to have a clinical efficacy of 70.6% in treating CDI, as determined by the absence of CDI diarrhea for 8 weeks after treatment. See, Khanna, et al., Drugs 2022; 82: 1527-1538.

[0116] Additionally or alternatively, CDI treatment success may be assessed with reference to a sustained clinical response for 6 months. For instance, treatment success may be assessed with reference to the absence of CDI diarrhea (passage of > 3 unformed / loose stools [i.e., Bristol Stool Scale type 6-7] in 24 consecutive hours for > 2 consecutive days) for a period of time, such as for 6 months after treatment. Thus, in some embodiments, the patient does not experience CDI diarrhea for at least 6 months following treatment with a microbiota restoration therapy composition as described herein. In some embodiments, efficacy of a method as described herein is at least non-inferior to a method comprising administering the microbiota restoration therapy composition by enema.

[0117] On the other hand, if a patient meets criteria for treatment failure, the patient may receive a second course of the microbiota restoration therapy, such as within 21 calendar days of confirmed failure. Treatment failure (CDI recurrence) may be defined as the presence of CDI diarrhea within 8 weeks of administration of microbiota restoration therapy. If antibiotics were given to control symptoms, a 24 to 72 hour washout period may be required prior to administration of the second course of the microbiota restoration therapy.4. Improvement of Quality of Life

[0118] CDI and recurrent CDI (rCDI) may lead to markedly reduced health-related quality of life (HRQoL), such as relating to psychological and emotional well-being, and may include feelings of embarrassment, anxiety, and depression. Fear and concerns about worsening CDI, recurrence and transmitting disease to others may lead to feelings of social isolation, loneliness and worry of hospitalization. For example as reported in Example 3, patients with rCDI report having one or more of needing proximity to bathroom facilities, having impacted leisure time, having disrupted sleep, having a reduced ability to eat nutritious meals, fearing rCDI will not resolve, and having debilitating fatigue.

[0119] Health-related quality of life (HR-QoL) in patients with recurrent CDI may be assessed using the C. t / / Health-Related Quality-of-Life (Cdiff32) questionnaire. As seen in FIG. 11, theAtty. Docket No. 052209-0664Cdiff32 questionnaire includes 32 self-reported questions across physical, mental and social domains that focus on the impact of CDI. Each question has five responses evaluated using the Likert-scale indicating the degree to which an individual’s CDI disease has impacted their everyday life during the past 7 days. Questions that relate to the physical domain relate to daily activities, diet, sleep, and discomfort. Questions that relate to the mental domain relate to anxiety, coping with disease / health perception, control of disease, impact of stress and dysphoria. Questions that relate to the social domain relate to relationships and social reaction.

[0120] Treatment with a microbiota restoration therapy composition as described herein may improve quality of life, such as may be assessed by the C. diff Health-Related Quality-of-Life (Cdiff32) questionnaire. The improvement in quality of life may relate to one or more of the physical, mental and social domains. In some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients experience an improvement in quality of life relating to the physical domain, such as one or more relating to daily activities, diet, sleep, and discomfort. Additionally or alternatively, in some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients experience an improvement in quality of life relating to the mental domain, such as one or more relating to anxiety, feeling irritable and / or isolated, fear of further diarrhea of antibiotics causing CDI again, and worry about transmitting CDI to family or friends.

[0121] In some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients experience an improvement in quality of life such as relating to one or more of anxiety, coping with disease / health perception, control of disease, impact of stress and dysphoria. In some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients experience an improvement in quality of life such as relating to one or more of relationships and social reaction. In some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients experience an improvement in quality of life such as relating to daily activities, diet, sleep, and discomfort.5. Treatment of Other Gastrointestinal Diseases or Disorders

[0122] Provided in accordance with another aspect of the present disclosure are methods for treating other gastrointestinal diseases or disorders by administering a dose of aMRT compositionAtty. Docket No. 052209-0664 as described herein to a patient in need thereof by colonoscopy or rectally (e.g., by enema). Examples of such other gastrointestinal diseases or disorders include, but are not limited to, irritable bowel syndrome or spastic colon, functional bowel disease (FBD), including constipation predominant FBD, pain predominant FBD, and upper abdominal FBD, non-ulcer dyspepsia (NUD), gastroesophageal reflux, inflammatory bowel disease including Crohn’s disease, ulcerative colitis, indeterminate colitis, collagenous colitis, microscopic colitis, pseudomembranous colitis, mucous colitis, antibiotic associated colitis, idiopathic or simple constipation, diverticular disease, AIDS enteropathy, small bowel bacterial overgrowth, celiac / coeliac disease, polyposis coil, colonic polyps, chronic idiopathic pseudo obstructive syndrome, chronic gut infections with specific pathogens including bacteria, viruses, fungi and protozoa; viral gastrointestinal disorders, including viral gastroenteritis, Norwalk viral gastroenteritis, rotavirus gastroenteritis, and AIDS related gastroenteritis. Additional information on the treatment of gastrointestinal diseases or disorders by administration of an MRT composition is disclosed in U.S. Patent No. 9,675,648, which is incorporated herein by reference in its entirety.

[0123] In specific embodiments, methods as disclosed herein are used to treat ulcerative colitis (UC) or Chron’s disease. In some embodiments, a method as described herein may be used to treat ulcerative colitis (UC) by administering a dose of an MRT composition as described herein to a patient in need thereof by colonoscopy. In some embodiments, a method as described herein may be used to treat Crohn’s disease (CD) by administering a dose of a MRT composition as described herein to a patient in need thereof by colonoscopy.

[0124] Ulcerative colitis is a relapsing and remitting disease characterized by acute non- infectious inflammation of the colorectal mucosa and is diagnosed and classified by colonoscopy and histological findings. Active episodes of UC (flares) are marked by passing of blood and mucus, diarrhea, and abdominal pain, frequently accompanied by urgency and tenesmus. Involvement of the rectal mucosa is a constant feature while the inflammation spreads to higher levels in a continuous and retrograde fashion, with no sparing of mucosa from the anorectal junction. Hence, UC may be divided into proctitis, proctosigmoiditis, left-sided colitis (the proximal limit being below the splenic flexure), extensive colitis (involving the transverse colon), and pancolitis (involving the entire colon). In the most severe forms, systemic signs comprising fever, loss of appetite, fatigue, night sweats, and weight loss may occur. In a given year,Atty. Docket No. 052209-0664 approximately 30% of people with UC in the US have mild disease activity, 20% have moderate disease activity, 1 to 2% of patients have severe disease, and 48% of patients are in remission. Despite treatment, it is estimated that approximately 70% of patients who have active disease in a given year will experience a relapse in the following year. Conversely, only 30% of patients in remission in a given year will experience a relapse in the following year. The longer a patient remains in remission, the less likely he or she is to experience a relapse (flare-up) of the disease in the following year.

[0125] The aim of treatment of active mild to moderate UC typically is induction and maintenance of remission, improvement in quality of life, and prevention of complications associated with long-term UC. The compositions and methods described herein may be effective for treating active mild to moderate UC as assessed by one or more of induction of remission, maintenance of remission, improvement in quality of life, and prevention or reducing the risks of complications associated with long-term UC, including one or more of induction of corticosteroid- free clinical remission with endoscopic remission, induction of corticosteroid-free clinical remission with endoscopic response, induction of clinical remission, induction of clinical response with endoscopic remission or clinical response with endoscopic remission, induction of endoscopic remission, and induction of endoscopic response.

[0126] Thus, some embodiments of treatment methods disclosed herein comprise administering a dose of a microbiota restoration therapy (MRT) composition as described herein to a subject in need of treatment of UC, such as a subject suffering from UC. In specific embodiments, the subject is a human suffering from UC. In further specific embodiments, the subject is an adult human suffering from UC. In further specific embodiments, the subject is an adult male human suffering from UC. In further specific embodiments, the subject is an adult female human suffering from UC. The UC treatment methods described herein may comprise, for example, administration of a dose of the MRT composition by colonoscopy. Additionally or alternatively, the UC methods of treatment may comprise a course of treatment, such as once weekly administration (e.g., rectally, such as by enema) over an 8-week course of treatment. Without being bound by theory, it is believed that the administered fecal microbes restore the diversity of the gut microbiota in patients with UC and decrease disease burden by normalizing abnormal immune and inflammatory responses.Atty. Docket No. 052209-0664

[0127] While not limited thereto, the methods of treating UC described herein may be particularly suited for subjects with mild to moderate UC. In some embodiments, the subject has an established diagnosis of UC by standard clinical, endoscopic and histological criteria. In some instances, the subject has mild to moderate UC as defined by a modified Mayo score > 4 and < 7, including an endoscopic subscore > 2 and a rectal bleeding subscore >1 at the start of treatment. The Mayo scoring system is the most commonly used scoring system for UC in clinical trials and routine practice and standardizes the severity of a patient’ s UC symptoms, which allows physicians to assess response to treatment over time. The Mayo scoring system includes four subscores: stool frequency, rectal bleeding, physician’s global assessment, and endoscopic findings, as illustrated in the table below.Table 1 Mayo Scoring System for the Assessment of Ulcerative Colitis ActivityAtty. Docket No. 052209-0664a Each patient serves as his or her own control to establish the degree of abnormality of the stool frequency. b The daily bleeding score represents the most severe bleeding of the day. c The physician’s global assessment acknowledges the 3 other criteria, the patient’s daily recollection of abdominal discomfort and general sense of well-being, and other observations, such as physical findings and the patient’s performance status.

[0128] The Mayo score ranges from 0 to 12, with higher scores indicating more severe disease. A modified Mayo score excludes the physician’s global assessment and ranges from 0 to 9. A partial Mayo score excludes endoscopy and ranges from 0 to 9. In some embodiments, a modified Mayo score may be used for assessment of a subject’s status prior to, during or after treatment, clinical remission, and / or response. In some embodiments, a partial Mayo score may be used for assessment of a subject’s status prior to, during or after treatment, and / or clinical remission and / or response.

[0129] In some embodiments, a subject may be assessed for one or more of the following statuses using the Mayo scoring system before, during, or after a course of treatment, and / or at any time thereafter: corticosteroid-free clinical remission with endoscopic remission or endoscopic response, clinical remission, clinical response with endoscopic remission or endoscopic response, and endoscopic remission or endoscopic response. As used herein, a subject is considered “corticosteroid free” if the subject is not using corticosteroids, which may be assessed at the end of MRT treatment or thereafter. The table below summarizes definitions for clinical and endoscopic remission and response. In the table below, the time points refer to specific assessment timepoints of the clinical trial outlined below, and are not limiting.Table 2. Definitions of Clinical and Endoscopic Remission and ResponseAtty. Docket No. 052209-0664

[0130] Thus, in some embodiments, a subject treated for UC as described herein exhibits a status after treatment, including after treatment for 8 weeks, as may be assessed after treatment, such as after treatment for 8 weeks, such as 8 weeks after starting treatment, or thereafter, including 52 weeks after starting treatment, selected from: corticosteroid-free clinical remission with endoscopic remission; corticosteroid-free clinical remission with endoscopic response; clinical remission; clinical response with endoscopic remission; clinical response with endoscopic response; endoscopic remission; and endoscopic response.

[0131] Thus, the compositions and methods described herein may be effective for treating active mild to moderate UC as assessed by one or more of induction of remission, maintenance of remission, improvement in quality of life, and prevention or reducing the risks of complications associated with long-term UC, including one or more of induction of corticosteroid-free clinical remission with endoscopic remission, induction of corticosteroid-free clinical remission with endoscopic response, induction of clinical remission, induction of clinical response with endoscopic remission or clinical response with endoscopic remission, induction of endoscopic remission, and induction of endoscopic response.Atty. Docket No. 052209-0664

[0132] The following embodiments of UC treatment methods are provided:Embodiment 1 : A method for treating ulcerative colitis in a human patient in need thereof, comprising administering a dose of a microbiota restoration therapy composition to the patient, wherein the microbiota restoration therapy composition comprises fecal microbes obtained from a human donor fecal sample. Embodiment 2: The method of embodiment 1, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising from about I x lO8to about 5 x IO10colony -forming units (CFU) / mL of the fecal microbes, and wherein the method comprises administering the composition rectally or by colonoscopy. Embodiment 3 : The method of embodiment 2, wherein the fecal microbiota suspension further comprises a solution comprising polyethylene glycol and saline, optionally wherein the polyethylene glycol is PEG 3350. Embodiment 4. The method of embodiment 2 or embodiment 3, wherein the dose of the composition has a volume of about 150 mb. Embodiment 5: The method of any one of embodiments 2-4, wherein fecal microbes present in the dose of the composition include >1x105 CFU / mL Bacteroides. Embodiment 6: The method of any one of embodiments 2-5, wherein the method comprises rectally administering the dose of the composition by enema once weekly for 8 weeks. Embodiment 7: The method of any one of embodiments 2-5, wherein the method comprises administering the composition by colonoscopy. Embodiment 8: The method of any one of the preceding embodiments, wherein the fecal microbes comprise microbes of species Bacteroides Embodiment 9: The method of any one of the preceding embodiments, wherein the ulcerative colitis is mild to moderate ulcerative colitis. Embodiment 10: The method of any one of the preceding embodiments, wherein the ulcerative colitis is mild to moderate ulcerative colitis as defined by a modified Mayo score > 4 and < 7 with an endoscopic subscore >2 and a rectal bleeding sub score >1. Embodiment 11 : The method of any one of the preceding embodiments, wherein the patient has completed a course of antibiotic therapy prior to treatment with the microbiota restoration therapy composition. Embodiment 12: The method of any one of the preceding embodiments, wherein the microbiota restoration therapy composition is first administered 24 to 72 hours after a last dose of the antibiotic. Embodiment 13: The method of any one of the preceding embodiments, wherein, after treatment with the microbiota restoration therapy composition, the patient is in corticosteroid-free clinical remission with endoscopic remission. Embodiment 14: The method of any one of the preceding embodiments, wherein, after treatment with the microbiotaAtty. Docket No. 052209-0664 restoration therapy composition, the patient is in corticosteroid-free clinical remission with endoscopic response. Embodiment 15: The method of any one of the preceding embodiments, wherein, after treatment with the microbiota restoration therapy composition, the patient is in clinical remission. Embodiment 16: The method of any one of the preceding embodiments, wherein, after treatment with the microbiota restoration therapy composition, the patient exhibits clinical response with endoscopic remission or clinical response with endoscopic remission. Embodiment 17: The method of any one of the preceding embodiments, wherein, after treatment with the microbiota restoration therapy composition, the patient is in endoscopic remission. Embodiment 18: The method of any one of the preceding embodiments, wherein, after treatment with the microbiota restoration therapy composition, the patient exhibits endoscopic response.6. Treatment of Cancers

[0133] Provided in accordance with another aspect of the present disclosure are methods for treating cancer by administering a dose of a MRT composition as described herein to a patient in need thereof by colonoscopy, or rectally. Without being bound by theory, it is believed that administration of the MRT composition may enhance the immune system of a patient who has cancer. Illustrative examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, melanoma, non-squamous cell lung cancer, squamous cell lung cancer, renal cell carcinoma, head and neck tumors, bladder cancer, Hodgkin lymphoma, nonHodgkin lymphoma, gastric cancer, colorectal cancer, multiple myeloma, esophageal cancer, breast cancer, glioblastoma, mediastinal B-cell lymphoma, other hematologic malignancies, testicular cancer, pancreatic cancer, lymphoma, cervical cancer, ovarian cancer, basal cell carcinoma, neuroblastoma, leukemia, and sarcoma. Other examples of cancer include gastrointestinal cancer, which include one or more of stomach cancer, esophageal cancer, colon cancer, gallbladder cancer, liver cancer, pancreatic cancer, colorectal cancer, anal cancer, bladder cancer, prostate cancer, and gastrointestinal stromal tumors. Additional information on the treatment of cancer by administration of an MRT composition is disclosed in US Pre-Grant Publication 2021 / 0283197, which is incorporated herein by reference in its entirety.

[0134] In some embodiments, methods as described herein may be used to treat colorectal cancer by administering a dose of a MRT composition as described herein to a patient in need thereof byAtty. Docket No. 052209-0664 colonoscopy. In some embodiments, methods described herein may be used to treat bladder cancer by administering a dose of a MRT composition as described herein to a patient in need thereof by colonoscopy.

[0135] In any embodiments of treating cancer, the methods may further comprise administration of a cancer therapeutic agent or other cancer therapy. Examples of cancer therapeutic agents and cancer therapies include, but are not limited to, immune checkpoint inhibitors, bispecific T-cell activating antibody agents, and adoptive T-cell transfer therapy. Examples of immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 agents, and the like. Such agents and therapies may be used in combination of one or more thereof. Without being bound by theory, it is believed that treatment with an MRT composition as disclosed herein in conjunction with immune checkpoint inhibitor immunotherapy (such as treatment with a PD-1 inhibitor) may enhance the efficacy of the immune checkpoint inhibitor immunotherapy, such as by enhancing local and / or systemic immune responses, such as by promoting antitumor T cell responses and / or inhibiting immunosuppression. See, e.g., Routy et al., Nature Medicine, 2023: 29: 2121-32; Di Luccia et al., Sci. Imunol., 2024, 9: eadi5374. For example, treatment with an MRT composition as disclosed herein may overcome resistance to immune checkpoint inhibitor immunotherapy that many cancer patients experience.

[0136] Additionally or alternatively, in any embodiments of treating cancer, the treatment may be effected in conjunction with surgical treatment of the cancer, such as surgery to remove a tumor or other cancerous tissue. Without being bound by theory, treatment with a MRT composition as described herein prior to surgery may significantly increase cytotoxic T cells and / or reduce regulatory T cells in both peripheral blood and tumors. The increase in cytotoxic T cells and / or reduction of regulatory T cells may enhance the efficacy of the cancer treatment. Additionally or alternatively, treatment with a MRT composition may modulate the metabolic function of regulatory T cells. In accordance with such embodiments, the MRT composition may be administered by colonoscopy prior to surgery, such as 1, 2, 3, or 4 weeks, or longer, prior to surgery. In some embodiments, the MRT composition is administered by colonoscopy two weeks prior to surgery. In other embodiments, the MRT composition is administered by colonoscopy four weeks prior to surgery.Atty. Docket No. 052209-06647. Treatment of Antimicrobial Resistance Colonization

[0137] Provided in accordance with another aspect of the present disclosure are methods for decolonizing patients that carry antibiotic resistance genes (ARGs), e.g., in their gut microbiota, e.g., to achieve antimicrobial resistance (AMR) gut decolonization, e.g., to reduce antimicrobial resistance gene abundance and / or reduce the presence of antimicrobial resistant organisms. Such methods comprise administering a dose of a microbiota restoration therapy composition as described herein to the patient rectally or by colonoscopy.

[0138] Antibiotic use remains a risk factor for recurrent Clostridioides difficile Infection (rCDI) due to disruption in gut microbiome composition and diversity. Many bacteria species resist antimicrobial compounds via diverse antimicrobial resistance (AMR) mechanisms, potentially leading to increased risks of loss of antimicrobial efficacy and colonization of multidrug-resistant organisms. Fecal microbiota transplantation and the administration of live biotherapeutic products (LBPs) may help to decolonize patients of bacteria that carry antibiotic resistance genes (ARGs). See, e.g., Durcamon QR, et al. J Infect Dis. 2021;223:S283-S289; Merrick B, et al. Infect Dis Rep. 2023;15:238-254). An MRT composition as described herein (now FDA-approved as Fecal microbiota, live-jslm (REB YOTA®) and previously referred to as RBX2660) was reported to reduce ARG abundance and resistant organisms in the recipient gut microbiome in a previous phase 2 trial assessing efficacy and safety, based on shallow shotgun sequencing. See Langdon A, et al. Genome Med. 2021 ; 13:28. As reported in Example 7 herein, a post-hoc analysis of data from the double-blind, placebo-controlled phase 3 trial referred to as PUNCH CD3 (NCT03244644), revealed that clinical efficacy against CDI was correlated with increases in the relative abundance of Bacteroidia and Clostridia, two bacterial classes associated with a healthy microbiome, as well as decreases in ARG abundance.

[0139] Without being bound by theory, it is believed that the administered fecal microbes repopulate the gut microbiota, restore diversity of the gut microbiome, and / or reduce ARG richness. As used herein, “ARG richness” is defined as the number of unique ARGs in a given sample. ARGs can be annotated with a hierarchical taxonomy from type (e.g., drugs, biocides, multicompound, metals) to class (e.g., aminoglycosides, beta-lactams, copper resistance) to mechanism (e.g., Class A beta-lactams, Class B beta-lactams), to group (e.g., CTX-M-15,Atty. Docket No. 052209-0664 extended-spectrum beta-lactamases (ESBL), A16S, AAC2-PRIME). See, e.g., Bonin N et al. Nucleic Acid Res. 2023; 5ED744-D752. Clinically important ARG classes include but are not limited to beta-lactam ARGs, fluoroquinolone ARGs, glycopeptide ARGs, polycationic peptide antimicrobial ARGs e.g., ARGs for polymyxin antibiotics such as colistin), and tetracyline ARGs. Clinically critical ARG groups include but are not limited to clinically critical gram-negative betalactam resistance-associated ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance-associated ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF), clinically critical colistin resistance-associated ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ), and clinically critical vancomycin-resistance- associated ARGs (such as VanA, VanHA, and VanXA).

[0140] Thus, in accordance with some aspects of the methods described herein, treatment with an MRT composition as described herein results in reduced richness of one or more clinically important or clinically critical ARG classes and / or one or more clinically important or clinically critical ARG mechanisms and / or one or more clinically important or clinically critical ARG groups, such as one or more clinically important ARG groups selected from gram-negative betalactam resistance-associated ARG groups, fluoroquinolone resistance-associated ARG groups, colistin resistance-associated ARG groups, and vancomycin-resistance-associated ARG groups, such as one or more selected from beta-lactam ARG richness, fluoroquinolone ARG richness, glycopeptide ARG richness, and colistin ARG richness, and / or one or more clinically critical ARG groups selected from clinically critical gram-negative beta-lactam resistance-associated ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance-associated ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF), clinically critical colistin resistance-associated ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ), and clinically critical vancomycin-resistance-associated ARGs (such as VanA, VanHA, and VanXA). In specific aspects, treatment with an MRT composition as described herein reduces beta-lactam ARG richness. In specific aspects, treatment with an MRT composition as described herein reduces fluoroquinolone ARG richness. In specific aspects, treatment with an MRT composition as described herein reduces beta-lactam ARG richness and fluoroquinolone ARG richness.Atty. Docket No. 052209-0664

[0141] In some aspects treatment with an MRT composition as described herein may increase tetracycline ARG richness (tetracycline resistance is prevalent in commensal bacteria in healthy populations).8. Altering Microbiome and Metabolome Composition

[0142] The gut microbiome includes a broad consortium of microbiota that, within the gastrointestinal environment and along with their collective functions, play an essential role in maintaining eubiosis and overall health and wellbeing. Commensal microbes such as Bacteroidetes and Firmicutes are the predominant bacterial phyla associated with gastrointestinal eubiosis. The healthy gut microbiome maintains overall health through regulating various metabolic pathways (e g., bile acid (BA) metabolism, complex carbohydrate digestion, and vitamin synthesis), supporting gut epithelial barrier functions, supporting immune system regulation, and providing protection against potential pathogens through colonization resistance and production of bacteriocins.

[0143] Gut microbiome dysbiosis, defined as disruption of the composition of the colonic microbiome and metabolome, permits infection and proliferation of opportunistic pathogens such as C. difficile. As many as 35% of patients treated for an initial CDI and up to 65% of patients with one or more prior CDI episodes develop recurrent CDI (rCDI). Antibiotics are the most common cause of the gut dysbiosis that can result in CDI. While antibiotics such as vancomycin and fidaxomicin are the standard-of-care (SOC) treatment for rCDI, they do not support microbiota restoration and, in the case of vancomycin, further potentiate gut dysbiosis and increase the risk of subsequent rCDI.

[0144] Treatment with a microbiota restoration therapy composition as described herein may result in the alteration or restoration of the gut microbiome. In some embodiments, after treatment with a microbiota restoration therapy composition as described herein, the relative abundance of one or more of Clostridia (e.g., Blautia, Lachnosclostridium, Faecalibacterium , Roseburia, and Eubacterium) and Bacteroidia (e.g., Bacteroides) in the patient’s gut may increase. Additionally or alternatively, in some embodiments, after treatment with a microbiota restoration therapy composition as described herein, the relative abundance of one or more of GammaproteobacteriaAtty. Docket No. 052209-0664(e ., Klebsiella, Escherichia, and Citrobacter), Bacilli (e.g., Lactobacillus and Streptococcus') and Negativi cutes in the patient’s gut may decrease.

[0145] In some instances, there may be a correlation with improved quality of life responses and specific microbiome changes. For example, after treatment with a microbiota restoration therapy composition as described herein, patients with improved quality of life responses (e.g., as assessed using the C. z / z / Health-Related Quality-of-Life (Cdiff32) questionnaire) may have an increase in the relative abundance of one or more of Clostridia (e.g., Blautia, Lachnosclostridium, Faecalibacterium, Roseburia, and Eubaclerium) and Bacteroidia (e.g., Bacteroides). Additionally or alternatively, after treatment with a microbiota restoration therapy composition as described herein, patients with improved quality of life responses (e.g., as assessed using the C. <7 / ( / Health- Related Quality-of-Life (Cdiff32) questionnaire) may have a decrease in the relative abundance of one or more of Gammaproteobacteria (e g., Klebsiella, Escherichia, and Citrobacter), Bacilli (e.g., Lactobacillus and Streptococcus) and Negativi cutes.

[0146] Treatment with a microbiota restoration therapy composition as described herein also may result in alteration or restoration of a metabolite metabolome, such as the gut bile acid (BA) metabolome or gut BA composition. Bile acids are the final product of cholesterol breakdown and function as a complex regulator and signaling factor in human metabolism. Bile acids are made exclusively in the liver in a series of enzymatic reactions in the hepatocyte that convert hydrophobic cholesterol into more water-soluble amphiphatic compounds, and the production of bile acids is localized primarily in the perivenous hepatocytes. Primary bile acids are the immediate products of the bile acid synthetic pathways and include cholic acid (CA), glycocholic acid (GA), taurocholic acid (TCA), and chenodeoxycholic acid. The action of intestinal bacterial flora on primary bile acids results in the formation of secondary bile acids, which include deoxycholic acid (DCA) and lithocholic acid (LCA).

[0147] In some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients may have an increased ratio of secondary bile acids to primary bile acids (S:P). Additionally or alternatively, in some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients may have an increased concentration of secondary bile acids (e.g., DCA and LCA). Additionally or alternatively, in some embodiments,Atty. Docket No. 052209-0664 after treatment with a microbiota restoration therapy composition as described herein, patients may have a decreased concentration of primary bile acids (e.g., GCA, CA, and TCA).

[0148] In some instances, there may be a correlation with improved quality of life responses and specific metabolome changes. For example, after treatment with a microbiota restoration therapy composition as described herein, patients with improved quality of life responses (e.g., as assessed using the C. / / / / Health-Related Quality-of-Life (Cdiff32) questionnaire) may have an increase in the ratio of secondary bile acids to primary bile acids (S:P), e.g., in the subject’s gut metabolome. Additionally or alternatively, in some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients with improved quality of life responses may have an increased concentration of secondary bile acids (e.g., DC A and LCA) , e.g., in the subject’s gut metabolome. Additionally or alternatively, in some embodiments, after treatment with a microbiota restoration therapy composition as described herein, patients with improved quality of life responses may have a decrease in the concentration of primary bile acids (e.g., GCA, CA, and TCA), e.g., in the subject’s gut metabolome.

[0149] Without being bound by theory, the strong association between improved quality of life responses and gut microbiome and metabolome composition indicates that the microbiota-gut- brain axis (GBA) link is modulated through the CNS, immune and gastrointestinal functions, such as involving indirect CNS modulation via the GBA. Gut microbiota, including Bacteroidia genera Bacteroides and Parahacteroides and Clostridia genera Clostridium and Ruminococcus, can synthesize neurotransmitters such as such as glutamate, gamma-aminobutyric acid, serotonin, norepinephrine, and dopamine, which act locally on the enteric nervous system. Additionally, there is cross-talk from the enteric nervous system back to the CNS, including via cytokine responses to enteric neural modulation. Microbiota-derived metabolites like short-chain fatty acids (SCFA) maintain intestinal epithelial barrier function, and neuroendocrine and immunologic homeostasis. Their depletion, or the depletion of Clostridia genera known to produce them, would likely alter circulating levels of microbial-derived neurotransmitters or cytokines that could alter CNS function. Several Clostridia and Bacteroidia genera are known to metabolize primary to secondary BA. Among other functions, secondary BAs like LCA inhibit C. difficile growth and toxin B expression, which could dampen toxin-triggered neuropeptide release and neurogenicAtty. Docket No. 052209-0664 inflammation. This may explain part of the secondary BA correlation with mental HRQOL score as measured by Cdiff32.9. Microbiota Restoration Therapy Compositions

[0150] A microbiota restoration therapy (MRT) composition suitable for use in the methods described herein typically is a liquid composition comprising human fecal microbiota (human fecal microbes). Typically, the human fecal microbes are obtained from human fecal matter (e.g., stool) sourced from a qualified human donor (e.g., a donor human fecal sample). Typically, the human donor is pre-screened for transmissible pathogens, such as one or more or all of Treponema, Hepatitis A, Hepatitis B, Hepatitis C, human immunodeficiency virus (HIV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Additionally or alternatively, a sample of the donor human fecal sample may be screened for transmissible pathogens before being used to prepare an MRT composition, or before the MRT composition is released for use. For example, donor human fecal samples may be screened for the presence of one or more or all of Clostridium species including C. difficile, Norovirus, Adenovirus, enteric pathogens, antigens to Giardia species, Cryptosporidia species and other pathogens, including acid-fast bacteria, enterococci, including but not limited to vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as for ova or parasitic bodies, including spore-forming parasites, including but not limited to Isospora, Cyclospora, and Cryptospora.

[0151] A MRT composition as described herein may be a liquid suspension of fecal microbes. For example, the composition may further comprise, in addition to the fecal microbes, polyethylene glycol and saline. In some embodiments, the polyethylene glycol comprises polyethylene glycol 3350. In some embodiments, the saline is a 0.9% solution of sodium chloride. The solution comprising polyethylene glycol and saline may comprise from 20 g to 50 g polyethylene glycol per 1 L of saline, such as 20, 25, 30, 35, 40, 45, or 50 g polyethylene glycol per 1 L of saline. In some embodiments, the solution comprises 30 g polyethylene glycol per 1 L of saline.

[0152] A dose of a MRT composition as described herein may be prepared from any suitable amount of donor human fecal sample. In some embodiments, a dose of a MRT composition as described herein is prepared from about 50 g to about 60 g of a donor human fecal sample, suchAtty. Docket No. 052209-0664 as about 50 g, about 51 , about 52 g, about 53 g, about 54 g, about 55 g, about 56 g, about 57 g, about 58g, about 59 g, or about 60 g of a donor human fecal sample.

[0153] A MRT composition as described herein may be prepared by any suitable process. For instance, an MRT composition may be manufactured from human fecal matter sourced from qualified donors, as described above. In some embodiments, a MRT composition as described herein is obtained by a process comprising processing a donor human fecal sample with a solution comprising, e.g., polyethylene glycol and saline (e.g., a solution as described above) and obtaining a filtrate comprising a suspension comprising fecal microbes. A suitable process for obtaining an MRT composition as described herein is set forth in U.S. Patent 9,675,648, for example. The MRT composition may be prepared under Good Manufacturing Practice (GMP) conditions. For instance, the MRT composition may be manufactured from human fecal matter sourced from qualified donors. As noted above, a sample of the donor human fecal matter typically is tested for a panel of transmissible pathogens. The MRT composition may be prepared from the filtrate generated by processing the donor human fecal matter in a predefined ratio with a solution of polyethylene glycol (e.g., PEG 3350) and saline (e.g., a solution as described above). For example, the MRT composition may be prepared by collecting a fresh human fecal sample (donor human stool); adding a solution of polyethylene glycol (e.g., PEG 3350) in saline to the fresh human fecal sample to form a diluted sample; mixing the diluted sample with a mixing apparatus (such as a paddle mixer); and filtering the diluted sample (optionally using a filter bag), wherein filtering forms a filtrate comprising a suspension comprising fecal microbes.

[0154] In some embodiments, a dose of a MRT composition as described herein comprises no more than about 6 grams of polyethylene glycol. In some embodiments, a dose of an MRT composition as described herein comprises no more than 5.97 grams of polyethylene glycol. In some embodiments, a 150 mL dose of an MRT composition as described herein comprises no more than 5.97 grams of polyethylene glycol in saline.

[0155] A dose of a MRT composition as described herein may comprise from about 150 g to about 192 g of the solution of polyethylene glycol in saline, such as about 150 g, about 155 g, about 160 g, about 165 g, about 170 g, about 175 g, about 180 g, about 185 g, about 190, or about 192 g of the solution of polyethylene glycol in saline. In some embodiments, a dose of the MRTAtty. Docket No. 052209-0664 composition comprises from about 150 g to about 192 g of the solution of polyethylene glycol in saline. In some embodiments, a 150 mb dose of the MRT composition comprises from about 150 g to about 192 g of the solution of polyethylene glycol in saline.

[0156] In some embodiments, a dose of the MRT composition comprises from about 1 x 108to about 5 x IO10colony -forming units (CFU) / mL of fecal microbes. In some embodiments, the fecal microbes comprise spore-forming microbes and non-spore forming microbes. In some embodiments, the fecal microbes comprises microbes of species Bacteroides. In some embodiments, a dose of the MRT composition comprises > IxlO5CFU / mL of Bacteroides.

[0157] A dose of the MRT composition may have a volume of from about 25 mb to about 250 mb. In some embodiments, a dose of the MRT composition has a volume of about 150 mb to about 170 mb. In some embodiments, a dose of the MRT composition has a volume of about 150 mb. In some embodiments, a 150 mb dose of the MRT composition includes 1 x 108to 5 x 1010CFU / mL of diverse spore-forming and non-spore-forming bacteria, including Bacteroides.

[0158] The MRT composition may be supplied in a sample bag, such as a sample bag having a spike port and a fill port (e.g., a sealed 250 mb ethyl vinyl acetate (EVA) bag container closure with a spike port and a fill port). FIG. 1 illustrates a suitable sample bag 12. Sample bag 12 may be formed from any suitable material, such as ethyl vinyl acetate. Additionally or alternatively, sample bag 12 may include a polyethylene terephthalate polyester film, a material substantially impervious to gases, other polymers, or the like. Sample bag 12 may resemble an intravenous bag. Sample bag 12 may include an attachment that will allow sample bag 12 to be hung on a stand, e.g. to be positioned / hung above an endoscope.

[0159] Sample bag 12 may have a capacity in the range of about 25-250 ml (e.g., 250 ml). Sample bag 12 may have a fill port 24 for filling the MRT composition into sample bag 12. Fill port 24 may include a luer or other type of adapter to facilitate filling of the MRT composition. After filling sample bag 12 with the MRT composition, fill port 24 may be sealed, effectively sealing the MRT composition within sample bag 12. Sample bag 12 may also include a spike port 26. Spike port 26 may be utilized to dispense the MRT composition from bag 12 at the time of use.Atty. Docket No. 052209-0664

[0160] In addition to the sample bag, a tube assembly comprising a tube body, a clamp, and a spike port adapter positioned at one end of the tube body may be provided for administration of the MRT composition. Typically, the tube assembly is provided unattached from the sample bag. FIG. 2 illustrates tube assembly 16 as an example of a suitable tube assembly. Tube assembly 16 may include a spike member 28 that is designed to pierce spike port 26 on sample bag 12. Tube assembly 16 may also include a tube body 30 with a step adapter 32. Step adapter 32 may allow tube 16 to be coupled with a delivery tube 34.

[0161] Tube assembly 16 may include a number of additional features. For example, a clamp 36 may be coupled to delivery tube 34. A visual marker 38 may be disposed along delivery tube 34. A spike cover 40 may be disposed along spike member 28.

[0162] The MRT composition may be formulated and packaged for administration colonoscopy. When the MRT composition is administered by colonoscopy, the composition may be administered to the right side of the colon, such as between the ileocecal valve and the hepatic flexure of the colon.

[0163] Alternatively, the MRT composition may be formulated and packaged for administration rectally. A dose of the MRT composition may be administered rectally by enema. In some embodiments, a dose of the MRT composition is administered rectally by enema for a course of treatment, such as once weekly for 8 weeks.

[0164] In some embodiments, the MRT composition is RBX2660 (approved for administration via enema as REBYOTA®). RBX2660 is an opaque fecal microbiota suspension for rectal administration and is manufactured from human fecal matter sourced from qualified donors. The human fecal matter is tested for a panel of transmissible pathogens. The fecal microbiota suspension is the filtrate generated by processing the fecal matter in a predefined ratio with a solution of polyethylene glycol (PEG) 3350 and saline (which may be prepared by adding 30 g polyethylene glycol 3350 to 1.0 L saline). Each 150mL dose of RBX2660 contains between IxlO8and 5xl010colony forming units (CFU) per m of fecal microbes, including >lxl05CFU / mL of Bacteroides, and contains not greater than 5.97 grams of PEG3350 in saline. RBX2660 may be supplied as a pre-packaged single-dose (150 mL), in an ethyl vinyl acetate (EVA) bag fitted with a spike port.Atty. Docket No. 052209-0664

[0165] The MRT compositions may have a microbial colony forming unit (CFU) count of from about 30 CFU to about 300 CFU at a serial dilution of 10'6when cultured on Center for Disease Control (CDC) culture plate (e.g., CDC anaerobe 5% blood agar plate) or a microbial colony forming unit (CFU) count of from about 30 to about 300 CFU at a serial dilution of 10’5when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate. In some embodiments, a dose of the MRT composition has a microbial colony forming unit (CFU) count of from about 30 CFU to about 300 CFU at a serial dilution of 10‘6when cultured on a Center for Disease Control (CDC) culture plate. In some embodiments, a dose of the MRT composition has a microbial colony forming unit (CFU) count of from about 30 to about 300 CFU at a serial dilution of 10'5when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate.

[0166] Additionally or alternatively, the MRT composition may have a microbial colony forming unit (CFU) count of at least about 10 CFU per plate at a serial dilution of 104when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate or a microbial CFU count of from about 2.0 x 107CFU per mL to about 5.0 x 1010CFU per m when cultured on a Center for Disease Control (CDC) culture plate e.g., CDC anaerobe 5% blood agar plate). In some embodiments, the MRT composition has a microbial CFU count of at least about 10 CFU per plate at a serial dilution of 104when cultured on a BBE culture plate. In some embodiments, the MRT composition has a microbial CFU count of from about 2.0 x 107CFU per mL to about 5.0 x 1010CFU per mL when cultured on a CDC culture plate (e.g., CDC anaerobe 5% blood agar plate).

[0167] The plating tests referenced above can also be used to confirm the diversity of the living microbes present in the MRT composition. CDC plates and BBE plates, used individually or in combination, provide a way of assessing quality through diversity as described herein. For example, CDC plates can be designed to grow 5 to 7 different families of microbes that can be present in an MRT composition as described herein. BBE plate grows multiple species of the Bacteroides genus of microbes. Thus, threshold CFU counts on BBE plates indicate diversity within the Bacteroides genus present in the MRT composition.

[0168] The phenotypic diversity of the MRT compositions described herein may be determined by visually counting unique bacterial colonies based on morphological differences when appropriately MRT composition is plated on a culture plate, such as a Center for Disease ControlAtty. Docket No. 052209-0664(CDC) agar plate. In some embodiments, the MRT composition has bacteria from at least three colony types when cultured on a Center for Disease Control (CDC) culture plate (e.g., CDC anaerobe 5% blood agar plate).

[0169] MRT compositions as described herein may include bacteria that are members of at least 1 phylum, at least 2 phyla, at least 3 phyla, at least 4 phyla, at least 5 phyla, at least 6 phyla, at least 7 phyla, at least 8 phyla, at least 9 phyla, or at least 10 phyla. In some embodiments, an MRT composition as described herein includes bacteria that are members of at least 1 class, at least 2 classes, at least 3 classes, at least 4 classes, at least 5 classes, at least 6 classes, or at least 7 classes. In some embodiments, a MRT composition as described herein includes bacteria that are members of at least 1 order, at least 2 orders, at least 3 orders, at least 4 orders, at least 5 orders, at least 6 orders, or at least 7 orders. In some embodiments, an MRT composition as described herein includes bacteria that are members of at least 1 family, at least 2 families, at least 3 families, at least 4 families, at least 5 families, at least 6 families, at least 7 families. In some embodiments, a MRT composition as described herein includes bacteria that are members of at least 5, at least 10, at least 20, or at least 30 different genera of bacteria. In some embodiments, a MRT composition as described herein includes at least 10, at least 50, at least 100, at least 200, at least 300, or at least 400 different species of bacteria.

[0170] For example, an MRT composition as described herein may include viable bacteria from 1 or more orders or 2 or more orders including, but not limited to, Bacteroidales and Clostridiales . In some embodiments, about 20-95%, or about 30-85%, or about 40-60% of the viable bacterial in the MRT composition is from the or der Bacteroidales. Additionally or alternatively, about 10- 85%, or about 20-60%, or about 30-40% of the viable bacterial in the MRT composition is from the order Clostridiales.

[0171] Additionally or alternatively, a MRT composition as described herein may include bacteria from 5 or more families, or about 6-12 families, or about 7-10 families. This may include bacteria from families Bacteroidaceae, Burkholderiales, Clostridiaceae, Clostridiales, Eubacteriaceae , Firmicutes, Lachnospiraceae, Porphyromonadaceae, Prevotellaceae, Rikenellaceae, Ruminococcaceae, and Streptococcaceae . In some embodiments, about 20-84%, or about 30-50%, or about 36-48% of the viable bacterial in the MRT composition is from theAtty. Docket No. 052209-0664 family Bacteroidacea . Additionally or alternatively, about 0.5-2% or about 1% of the viable bacterial in the MRT composition may be from the family Burkholderiales. Additionally or alternatively, about 1-10%, or about 1-8%, or about 2-7% of the viable bacterial in the MRT composition may be from the family Clostridiaceae . Additionally or alternatively, about 1-22%, or about 3-22%, or about 1-10%, or about 1-8%, or about 4-7% of the viable bacterial in the MRT composition may be from the family Clostridiales . Additionally or alternatively, about 1-10%, or about 1-9%, or about 4-8% of the viable bacterial in the MRT composition may be from the family Eubcicteriaceae . Additionally or alternatively, about 0.5-2% or about 1% of the viable bacterial in MRT composition may be from the family Firmicutes. Additionally or alternatively, about 0.5- 23%, or about 1-10%, or about 4-9% of the viable bacterial in the MRT composition may be from the family Lachnospiraceae . Additionally or alternatively, about 0.5-8%, or about 1-5%, or about 1-3% of the viable bacterial in the MRT composition may be from the family Porphyromonadaceae . Additionally or alternatively, about 0.5-2% or about 1% of the viable bacterial in the MRT composition may be from the family Prevotellaceae . Additionally or alternatively, about 1-30%, or about 1-52%, or about 4-23% of the viable bacterial in the MRT composition may be from the family Rikenellaceae . Additionally or alternatively, about 5-30%, or about 8-25%, or about 10-18% of the viable bacterial in the MRT composition may be from the family Ruminococcaceae . Additionally or alternatively, about 0.5-2% or about 1% of the viable bacterial in the MRT composition may be from the family Streptococcaceae .

[0172] A MRT composition as described herein may have a Shannon Diversity Index of about 0.4-2.5, or about 1.0-2.0, or about 1.08-1.89, or about 1.25-1.75. These numbers are calculated at the “family” level. Doing calculations at other levels (e.g., phyla, species, etc.) would result in different numbers (e.g., 1-8 or so). Therefore, the Shannon Diversity Index may be on the order of about 1-8 when calculated at the phyla, species, or other levels.

[0173] In some embodiments, the MRT composition has a microbial colony forming unit (CFU) count of at least about 10 CFU per plate at a serial dilution of 104when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate. Additionally or alternatively, the MRT composition has a microbial colony forming unit (CFU) count of from about 2.0 x 107CFU per mL to about 5.0 x 1010CFU per mL when cultured on a Center for Disease Control (CDC) culture plate (e.g., CDC anaerobe 5% blood agar plate). Additionally or alternatively, the MRT composition has bacteriaAtty. Docket No. 052209-0664 from at least three colony types when cultured on a Center for Disease Control (CDC) culture plate (e.g, CDC anaerobe 5% blood agar plate).EXAMPLES

[0174] The following specific examples are included as illustrative of the compositions and methods described herein. These examples are in no way intended to limit the scope of the disclosure. Other aspects of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.Example 1: Retrospective Subgroup Analysis Of RBX2660 Administered By Colonoscopy Under Enforcement Discretion For Reduction Of rCDl

[0175] RBX2660 is a single dose, rectally administered, microbiota-based live biotherapeutic product (LBP) being investigated to reduce rCDI in adults. This example reports a retrospective subgroup analysis of the efficacy and safety of RBX2660 administered via colonoscopy under FDA enforcement discretion (ED). A primary objective of the retrospective study was to evaluate the safety and tolerability of RBX2660 through 6 months after treatment in patients who received RBX2660 under ED.

[0176] A full set analysis (FAS) was performed on data collected from patients >18 years of age who received RBX2660 between November 1, 2015, and September 30, 2019. Laboratory diagnosis of C. difficile was not required, and there were no exclusion criteria as long as the patient was eligible for treatment under ED. The treating site physician determined a patient’s eligibility to receive RBX2660 by colonoscopy. The primary endpoint was the number of patients with investigational product- and / or procedure-related treatment-emergent adverse events (TEAEs), defined as adverse events occurring on or after the day of treatment with RBX2660. Other safety endpoints included the number of adverse events per patient, number of TEAEs of special interest according to study definition, relatedness of TEAEs, severity of TEAEs, and onset of new chronic conditions relative to treatment administration or worsening of preexisting conditions. Treatment success, defined as the absence of CDI recurrence within 8 weeks after the final RBX2660 dose, and sustained clinical response for 6 months after treatment were secondary outcomes.Atty. Docket No. 052209-0664

[0177] A full analysis set (FAS) included all patients who met eligibility criteria and received RBX2660 during the study period. The FAS comprised 94 patients, of which 39 received one dose of RBX2660; the remaining patients received 2 or 3 doses of RBX2660 via rectal administration. Ten of the 39 patients who received one dose of RBX2660 for the qualifying event received the product via colonoscopy. Treatment success was achieved by 80% (n=8 / 10) of patients treated with RBX2660 administered by colonoscopy; 2 colonoscopy patients were considered treatment failures due to a lack of comprehensive records up to 6 months. Of the 8 patients who received RBX2660 via colonoscopy with treatment success at 8 weeks, 6 reported sustained clinical response at 6 months.

[0178] TEAEs of special interest included gastrointestinal adverse events, fever, chills, fatigue, possibility of disease transmission from donor to recipient, and adverse events related to the administration procedure. TEAEs were reported in 6 of 8 colonoscopy patients with comprehensive records; most were mild and moderate in severity, with gastrointestinal disorders and infections / infestations being the most common. Table 3 summarizes the adverse events in rCDI patients treated with RBX2660 administered by colonoscopy. One patient was hospitalized twice for 2 separate episodes of ileus. None of the TEAEs were considered related to RBX2660 or its administration.Table 3. Adverse Events in rCDI Patients Treated with RBX2660 Administered by Colonoscopy Under FDA Enforcement Discretion (6 Month Follow-up)Atty. Docket No. 052209-0664AE, adverse event“Event unrelated to CDI

[0179] The results of this retrospective analysis show that patients who received RBX2660 administered by colonoscopy have efficacy and safety profiles consistent with previous clinical trials of RBX2660 administered by enema, thus validating colonoscopy as a mode for MRT administration for reduction of rCDI.Example 2: A Multi-Center, Single-Arm Trial Exploring the Safety and Clinical Effectiveness of RBX2660 Administered by Colonoscopy to Adults with rCDI

[0180] The purpose of the multi-center, single arm clinical trial detailed in this example (CDI- SCOPE) is to explore the safety and clinical effectiveness of RBX2660 administered by colonoscopy to adults with rCDI. RBX2660 is provided as a single-dose preparation of a 150 mL liquid microbiota suspension in an ethylene vinyl acetate (EVA) bag fitted with a spike port. Each RBX2660 microbiota EVA bag contains 150 mL of a suspension of 1 x 108to 5 * 1010CFU / mL fecal microbes in a solution of polyethylene glycol 3350 and 0.9% Sodium Chloride Irrigation, US Pharmacopoeia (USP). One RBX2660 treatment course consists of the content of one RBX2660 microbiota EVA bag, delivered by colonoscopy.

[0181] Recurrent CDI (rCDI) for trial entry was defined as a documented diagnosis of (1) CDI diarrhea: the passage of >3 unformed / loose stools in 24 consecutive hours for >2 consecutive days, that began within 8 weeks after completion of previous CDI treatment and (2) >1 stool test positive for toxigenic C. difficile or C. difficile toxin, documented at the time of the CDI diarrhea for the qualifying CDI episode.

[0182] The recurrence of CDI during the trial, i.e., treatment failure, was defined as the presence of CDI diarrhea within 8 weeks after RBX2660 treatment. CDI diarrhea during the trial wasAtty. Docket No. 052209-0664 defined as passage of >3 unformed / loose stools (i.e., Bristol Stool Scale type 6-7) in 24 consecutive hours for >2 consecutive days and a stool test collected before start of any antibiotic treatment, which is positive for C. difficile toxin, as determined by a toxin protein detecting assay at the time of the diarrhea.

[0183] The primary endpoint of this study is RBX2660-related treatment-emergent adverse events (TEAEs) after RBX2660 treatment delivered by colonoscopy through 8 weeks, or treatment failure.

[0184] The secondary endpoints of this study were as follows:• Recurrence of CDI within 8 weeks after RBX2660 treatment by colonoscopy.• Time to CDI recurrence from baseline through 8 weeks after RBX2660 treatment by colonoscopy.• Physician-experience, as determined by questionnaire, documenting subjective experience of investigators on usability of RBX2660 in clinical practice when delivered by colonoscopy.• Physician perception of patient benefit, as determined by Clinician Global Impression of Improvement (CGI-I) at 8 weeks, or at treatment failure, after RBX2660 treatment by colonoscopy.• Patient-experience interview at 8 weeks, or at treatment failure, after RBX2660 treatment by colonoscopy.• Safety up to 6 months after RBX2660 treatment by colonoscopy:- TEAEs, including type, intensity, and causality.- Serious adverse events (SAEs).- Adverse events of special interest (AESIs): septic shock, toxic megacolon, colonic perforation, and emergency colectomy, and new onsets of obesity, glucose intolerance, metabolic syndrome, or autoimmune conditions.- Adverse events leading to death or intensive care unit (ICU) admission.Methodology

[0185] This study is a multi-center, single-arm clinical trial to explore the safety and clinical effectiveness of RBX2660 (human fecal microbiota suspension) when administered by colonoscopy to adults with rCDI. FIG. 3 illustrates the overall trial design of this study.Atty. Docket No. 052209-0664

[0186] Subjects aged 18 years and above, with a diagnosis of rCDI and a stool test positive for the presence of toxigenic C. difficile or C. difficile toxin for the qualifying rCDI episode, were eligible for screening in this trial. After signing the informed consent form (ICF), subjects underwent a screening evaluation prior to treatment at baseline (day 1). To be screened in this trial, subjects must have been taking or have just received a prescription for a course of antibiotics to control rCDI symptoms (type and dosage at the discretion of the prescribing physician). This means that subjects who had already completed their prescribed course of antibiotics to treat rCDI were not eligible for screening unless they had another recurrence that required treatment with antibiotics. Antibiotic therapy for the qualifying rCDI episode must have included a minimum of 10 consecutive days and must not have exceeded a total of 30 days (including intermittent dosing / pulse) of antibiotic use before a mandatory antibiotic washout period could be initiated. The antibiotic washout period consisted of a minimum of 1 day (>24 hours) to a maximum of 3 days (<72 hours) immediately before treatment at baseline. Before the antibiotic washout period could start, CDI symptoms must have been under control, i.e., no longer meeting the symptomatic criteria for CDI diarrhea, for the 2 consecutive days immediately before the antibiotic washout period. The CDI symptoms must furthermore have been under control throughout the antibiotic washout period and up to start of a mandatory bowel preparation (by a method at the investigator’ s discretion), in order for subjects to receive treatment with RBX2660. Any consented subjects who did not meet the eligibility criteria would have been considered as screening failures.

[0187] At the screening visit, subjects were instructed on how to document stool patterns in an electronic stool diary. Subjects were required to start completing the stool diary immediately after the screening visit, on the same day, and continue until start of bowel preparation. The stool diary was reviewed on an ongoing basis by trial site staff and at the baseline visit before administration of RBX2660. In order for subjects to receive treatment with RBX2660, the stool diary completed from the screening visit up to start of bowel preparation must demonstrate that CDI symptoms were under control, i.e., no longer meeting the symptomatic criteria for CDI diarrhea, for the 2 consecutive days immediately before initiating the antibiotic washout period, and throughout the antibiotic washout period up to start of bowel preparation. At baseline, immediately after the antibiotic washout period and bowel preparation, subjects received treatment with RBX2660,Atty. Docket No. 052209-0664 administered by colonoscopy. RBX2660 was to be administered by colonoscopy to the right side of the colon (i.e., between the ileocecal valve and the hepatic flexure of the colon).

[0188] Follow-up visits were to be conducted at 1, 2, 4, and 8 weeks after treatment, and 3 and 6 months after treatment. The follow-up visits at 1, 2 and 4 weeks and 3 months after treatment may be either virtual or on-site, as judged by the investigator. An on-site CDI recurrence visit would be required if CDI recurrence (treatment failure) is suspected at any time within 8 weeks after treatment with RBX2660. CDI recurrence is determined by use of an algorithm relating to diarrhea and C. difficile toxin test results from a toxin protein detecting assay. External adjudication is not used.

[0189] Subjects who completed the follow-up 4 visit at 8 weeks after RBX2660 treatment, or who were documented as treatment failures (recurrence of CDI) at a CDI recurrence visit, were contacted for a patient-experience interview by telephone.

[0190] According to the study protocol, subjects who are documented treatment failures, defined as presence of CDI diarrhea, including a stool test collected before start of any antibiotic treatment, which is positive for C. difficile toxin as determined by a toxin protein detecting assay, within 8 weeks after RBX2660 treatment would be treated according to standard of care (SoC) at the investigator’s discretion. These subjects would continue the follow-up assessments for safety through 6 months after RBX2660 treatment according to the trial flow chart. Any subjects who experience CDI after the follow-up visit at week 8 may be diagnosed and treated according to SoC at the investigator’s discretion. These subjects may continue the follow-up assessments for safety through 6 months after RBX2660 treatment according to the trial flow chart.

[0191] Adverse events, safety laboratory variables (clinical chemistry and hematology), vital signs, and concomitant medications will be assessed throughout the trial.

[0192] According to the study protocol, approximately 40 subjects were planned to receive treatment with RBX2660.Atty. Docket No. 052209-0664Inclusion Criteria

[0193] According to the study protocol, subjects must have met all of the criteria listed below to be eligible for participation:1. Signed informed consent obtained before any trial-related procedures at screening.2. Subject aged >18 years old at screening.3. Medical record documentation of recurrent Clostridioides difficile infection (rCDI) per the trial definition that includes >1 recurrence after a primary C. difficile infection (CDI) episode, at screening.4. Stool test positive for the presence of toxigenic C. difficile or C. difficile toxin for the qualifying rCDI episode, documented at the time of CDI diarrhea, at screening.5. Eligible for fecal microbiota transplantation (FMT) as judged by the investigator or current treatment guidelines for rCDI in the United States (US).6. Candidate for colonoscopy as judged by the investigator.7. Currently taking or have just received a prescription for a course of antibiotics to ensure control of CDI-related diarrhea, at screening. The antibiotic course should be for a minimum of 10 consecutive days, and a maximum of 30 days in total (including intermittent dosing / pulse) before the antibiotic washout period.8. Adherence to at least one of the following conditions throughout the trial period:The subject, if female, must: a) be post-menopausal (women >45 years with no menstrual period for >12 months without an alternative medical cause), or b) have been surgically sterilized, or c) have had a hysterectomy prior to screening, or d) use an adequate method of contraception (i.e., implants, injectables, hormonal intrauterine devices, combined hormonal contraceptives, having a vasectomized sexual partner, or total abstinence from heterosexual relations with no plans of becoming pregnant through insemination or in vitro fertilization).The subject, if male, must: a) use a non-hormonal single-barrier contraception (i.e., condom), and b) use an adequate method of contraception if his female partner is of childbearing potential (i.e., not post-menopausal) as defined above.This is however not required if the male subject is documented surgically sterile, remains sexually abstinent, when this is in line with his preferred and usual lifestyle, or if he has a female partner who is surgically sterilized, had a hysterectomy, or is post-menopausal.Atty. Docket No. 052209-06649. Willing to abstain from consuming probiotics (including over-the-counter and prescription) from screening through 8 weeks after RBX2660 treatment.10. Willing to comply with trial procedures, including attending scheduled visits, completion of the electronic stool diary, providing stool samples for future exploratory research, and adherence to treatment plan.Exclusion Criteria

[0194] According to the study protocol, subjects meeting any of the criteria listed below were not eligible for participation:1. Use or planned use of systemic antibiotics for an indication other than the qualifying rCDI episode.2. Current disease symptoms (e.g., diarrhea) caused by a confirmed intestinal pathogen other than C. difficile.3. Current uncontrolled chronic diarrhea not related to CDI.4. Current refractory CDI, i.e., CDI diarrhea not improving with antibiotics used to treat CDI.5. Fecal microbiota transplantation, treatment with other microbiota-based therapies, or treatment with RBX2660 other than the IMP: a) within 6 months before screening, or b) between screening and baseline.6. Receipt of CDI vaccine or treatment with CDI monoclonal antibodies: a) within 12 months before screening, or b) between screening and baseline.7. Compromised immune system including, but not limited to, inherited / primary and acquired immune disorders as judged by the investigator.8. Solid-organ transplant recipient.9. Initiation or escalation of systemic immunosuppressive agents, at the discretion of the investigator, for any condition: a) during the 8 weeks before screening, or b) between screening and baseline. Subjects on a stable dose of systemic immunosuppressants (e.g., <20 mg / day prednisone equivalent daily dose) may be considered eligible at the discretion of the investigator.10. Current or planned therapy that may cause diarrhea.11. Current or planned systemic cancer treatment (e.g., chemotherapy, radiotherapy, other).Atty. Docket No. 052209-066412. Diagnosis of short bowel syndrome.13. Current uncontrolled gastrointestinal motility disorders.14. Evidence of active, severe, or fulminant colitis.15. Diagnosis of toxic megacolon.16. Have a current colostomy or ileostomy.17. Major gastrointestinal tract surgery (not including appendectomy or cholecystectomy): a) within 60 days before screening, or b) between screening and baseline.18. Planned surgery requiring perioperative antibiotics.19. Life expectancy of <6 months, as judged by the investigator at screening.20. Current abuse of alcohol or drugs, as judged by the investigator at screening.21. Pregnant (as confirmed by a positive pregnancy test), breastfeeding, or intending to become pregnant, during trial participation.22. Participation in any interventional clinical trial during the past 12 weeks before screening.23. Known or suspected hypersensitivity to polyethylene glycol 3350.24. Severe food allergy, characterized as previous episodes of severe allergic reactions in the form of anaphylaxis after intake of food allergens.25. Inability to participate in the trial for other reasons, as judged by the investigator at screening.Duration Of Treatment

[0195] One RBX2660 treatment course consists of the content of one RBX2660 microbiota EVA bag, delivered by colonoscopy. RBX2660 was to be administered by colonoscopy to the right side of the colon (i.e., between the ileocecal valve and the hepatic flexure of the colon). Subjects were followed for 8 weeks after treatment for safety and CDI recurrence, and will be followed for up to 6 months after treatment for safety.Interim Results

[0196] The trial recruited 41 patients from across 12 trial sites, in 11 U.S. states. RBX2660- related treatment-emergent adverse events (TEAEs) after RBX2660 treatment delivered by colonoscopy through 8 weeks, or treatment failure have been recorded.Atty. Docket No. 052209-0664

[0197] Of the reported 57 TEAEs, 51 were considered not related to RBX2660. Of the 57 TEAEs, most were considered mild (42), 10 were moderate, 3 were potentially life-threatening, and 2 were severe. Constipation was the most common TEAE (6), followed by diarrhea (3) and abdominal pain / cramping (2). Three serious adverse events (SAEs) were reported (e.g., abdominal pain, brain neoplasm, and ulcerative colitis), none of which was deemed to be related to RBX2660. The 6 TEAEs considered to be related to RBX2660 were non-serious: abdominal distention (2), diarrhea, constipation, blood in stool, and hydration.

[0198] To date, 37 of the 41 treated patients had at least one day entered in the solicited events diary. All solicited events reported were primarily of mild-moderate severity; two patients reported severe events (increased diarrhoea of 1-day and 8-day duration, respectively). No serious adverse events were reported for solicited events.

[0199] To date, 28 of the 41 patients have reached the 8 week post-treatment mark for assessment of clinical effectiveness (treatment success). Treatment with RBX2660 was effective in all 28 patients (100%) with none of the patients experiencing recurrence of C. diff infection within 8 weeks of administration of RBX2660.

[0200] Physician-experience, as determined by a questionnaire documenting subjective experience of investigators on the usability of RBX2660 in clinical practice when delivered by colonoscopy was favorable, with mostly positive to very positive experience reported for all questions. Physician perception of patient benefit, as determined by the Clinician Global Impression of Improvement (CGI-I) assessment at 8 weeks (or at treatment failure, if applicable) was positive after RBX2660 treatment delivered by colonoscopy. To date, 35 CGI-I Scores have been completed for patients 8 weeks post treatment, with 11 being “much improved” and 24 being “very much improved.”

[0201] Patient-experience interviews were conducted with 15 patients at 8 weeks after RBX2660 treatment delivered by colonoscopy. As discussed in more detail in Example 3 below, posttreatment participants reported that diarrhea had either resolved completely (53.3%) or was less severe (46.7%), and described health-related quality of life improvements.Atty. Docket No. 052209-0664

[0202] Overall, these interim results indicate that RBX2660 is safe and effective at preventing recurrence of C. diff when administered by colonoscopy. Clinical efficacy for patient who have reached the 8 week post -treatment mark to date is 100%.Example 3: Improvement in Health-Related Quality of Life in Adults with rCDI after RBX2660 Administration by Colonoscopy

[0203] As noted above, the multi-center, single-arm Phase 3b clinical trial CDI-SCOPE was conducted to explore the safety and clinical effectiveness of RBX2660 administered by colonoscopy to adults with rCDI. All trial participants were invited to take part in a 1-hour interview exploring the burden of rCDI on HRQoL and any positive improvements in HRQoL after treatment. As noted above, patient-experience interviews have been conducted with 15 patients at 8 weeks after RBX2660 treatment delivered by colonoscopy. A trained interviewer conducted interviews using web-assisted software. Audio recordings were transcribed verbatim and de-identified prior to analysis.

[0204] Data on the 15 interview participants to date are presented in this interim analysis. The below table summarizes the participant characteristics. All 15 participants (100.0%) completed the 8-week follow-up; none experienced CDI recurrence. Before receiving RBX2660, most reported diarrhea as the most bothersome (66.7%) and the most important symptom (70.0%) to resolve. Participants reported that CDI impacted their day-to-day life (need proximity to bathroom: 80.0%; impacted leisure time: 80.0%; disrupted sleep: 73.3%; ability to eat nutritious meals: 73.3%), affected mental health (fear rCDI would not resolve: 60.0%), and led to debilitating fatigue (60.0%). Post RBX2660 treatment, participants reported diarrhea had either resolved completely (53.3%) or was less severe (46.7%). Average patient-reported diarrhea severity ratings [0-10, no symptoms to worst possible symptoms] dropped from 8.4 (SD: 1.6) before treatment to 0.3 (SD: 0.5) after. Participants described HRQoL improvements (improved physical function: 73.3%: ability to spend time with family again: 73.3%; able to return to leisure activities: 66.7%; feeling better mentally: 66.7%). This interim analysis indicates that RBX2660 treatment improves and / or completely resolves diarrhea symptoms and positively impacts key aspects of HRQoL and daily functioning.Atty. Docket No. 052209-0664Table 4. Participant Demographic and Clinical Characteristics (CDI-SCOPE Interim Analysis)Patient Characteristics Interview Population (N = 15)SexFemale 12 (80.0%)Male 3 (20.0%)Age (at time of interview)Mean (SD) 66.1 (14.6)Median (Range) 66.1 (37.7-85.6)RaceWhite 15 (100.0%)Black or African American 0 (0.0%)American Indian or Alaska Native 0 (0.0%)Asian 0 (0.0%)Native Hawaiian or Other Pacific Islander 0 (0.0%)EthnicityHispanic / Latino 0 (0.0%)Not Hispanic or Latino 15 (100.0%)NMore than 5 prior episodes 2 (14.3)SD: Standard deviation* n=l missing, thus N=14 for this characteristic.[1] Categorical variables are described by the frequency and percentage.[2] Continuous variables are described by their frequency, mean, standard deviation, median, and range (minimum and maximum values).Example 4: Administration of RBX2660 By Colonoscopy to Adults with UC or CD

[0205] Treatment with a MRT composition by colonoscopy as described herein may be used restore disrupted gut bacteria flora in patients suffering from UC or CD. A dose of a MRT composition as disclosed herein, such as RBX2660, will be administered by colonoscopy to patients suffering from UC or CD in a manner similar to what is disclosed in Example 2. TreatmentAtty. Docket No. 052209-0664 with the MRT composition by colonoscopy will restore a healthy population of gastrointestinal microbial floral, and thereby improve the patient’s condition.Example 5: Treatment of UC in Adults By Rectal Administration of RBX2660

[0206] Adults with active mild to moderate UC who are on stable treatment according to standard of care will be treated with RBX2660 administered rectally and compared to placebo. The effects on clinical and endoscopic remission and response will be evaluated after 8 weeks of once weekly treatment. Subjects will be followed for 52 weeks after start of treatment.

[0207] For purposes of inclusion criteria for this trial, UC status is defined as an established diagnosis of ulcerative colitis (UC) by standard clinical, endoscopic and histological criteria, for more than 3 months at screening. Active mild to moderate UC is defined by a modified Mayo score > 4 and < 7, including an endoscopic subscore > 2 and a rectal bleeding subscore >1 at randomization.

[0208] Exploratory objectives include to explore the effect of RBX2660 versus placebo in subjects with active mild to moderate UC on: the use of corticosteroid treatment; the alteration and durability of change in fecal-associated microbiota; the alteration and durability of change in mucosal microbiota; changes in the fecal metabolome; clinical and endoscopic remission and response; the need for biologies and / or small molecules for treatment of UC; C-reactive protein (CRP) and fecal calprotectin (FCP) levels; histological disease activity; changes from baseline in blood immune cell populations, and also to assess the onset of action in resolving symptoms (rectal bleeding and stool frequency).

[0209] The primary endpoint of this study is treatment-emergent adverse events from baseline to week 52. Exploratory endpoints include:• Corticosteroid-free clinical remission with endoscopic remission or response at weeks 8 and 52• Change from baseline in the fecal-associated microbiota in taxonomic and functional profding, assessed by shotgun metagenomics sequencing at weeks 1, 2, 3, 4, 5, 6, 7, 8, 26, and 52Atty. Docket No. 052209-0664• Change from baseline in the mucosal microbiota in taxonomic and functional profiling, assessed by shotgun metagenomics sequencing at weeks 8, and 52• Change from baseline in fecal metabolites (short chain fatty acid [SCFA] profiles, fecal bile acid [BA] profiles, and fecal tryptophan metabolites) at weeks 1, 4, 8, 26, and 52• Corticosteroid-free clinical remission with endoscopic remission at weeks 8 and 52• Corticosteroid-free clinical remission with endoscopic response at weeks 8 and 52• Clinical remission at weeks 8, 26 and 52• Clinical response at weeks 8, 26 and 52• Endoscopic remission at weeks 8 and 52• Endoscopic response at weeks 8 and 52• Change from baseline in use of biologies and / or small molecules for treatment of UC up to weeks 8, 26, and 52• Changes from baseline in C-reactive protein (CRP) and fecal calprotectin (FCP) levels at weeks 1, 2, 3, 4, 5, 6, 7, 8, 26, and 52• Histological remission (Nancy histological index [NHI] <2) at weeks 8 and 52• Time to symptoms resolution defined as a rectal bleeding score of 0 and a stool frequency score <1• Time to symptoms resolution defined as a rectal bleeding score of 0• Time to symptoms resolution defined as a stool frequency score of <1• Changes from baseline in immune cell populations (e.g., Thl, Th2, Th 17, and Treg) in blood at weeks 1, 4, 8, 26, and 52

[0210] For weeks 8 and 52, clinical remission is defined as a modified Mayo score <2 with a stool frequency subscores <1 and no greater than baseline, a rectal bleeding subscore = 0, and a Mayo endoscopic subscore <1. (The modified Mayo score consists of the stool frequency, rectal bleeding, and endoscopic Mayo subscores.) For week 26, clinical remission is defined as a partial Mayo score <2 with a stool frequency subscore <1, a physician’s global assessment subscore <1, and a rectal bleeding subscore = 0. (The partial Mayo score consists of the stool frequency, rectal bleeding, and physician’s global assessment Mayo subscores.) Endoscopic remission is defined as a Mayo endoscopic subscore <1. Endoscopic response is defined as a >1 point reduction from baseline on the Mayo endoscopic subscore.Atty. Docket No. 052209-0664

[0211] For weeks 8 and 52, clinical response is defined as a >2 point reduction, and at least a 30% reduction, from baseline on the modified Mayo score, and a >1 point reduction from baseline on the rectal bleeding subscore or an absolute rectal bleeding subscore <1. For week 26, clinical response is defined as a >2 point reduction, and at least a 30% reduction, from baseline on the partial Mayo score, and a >1 point reduction from baseline on the rectal bleeding subscore or an absolute rectal bleeding subscore <1.

[0212] Mayo subscores for stool frequency and rectal bleeding will be obtained from the subject electronic diary and calculated based on daily scores during 7 days before the visit. If stool frequency is 1 at baseline, symptom resolution is defined as a stool frequency <1.Methodology

[0213] The trial is a randomized, double-blind, placebo-controlled trial. The trial comprises a total of 13 visits to the trial site: visit 1 (screening), visit 2 (randomization), visit 3 (baseline; day 1), visits 4 to 12 (weeks 1, 2, 3, 4, 5, 6, 7, 8 [end-of-treatment], and 26), and visit 13 (week 52; end-of-trial). Subjects must have active disease (UC) and be on stable treatment for management of active UC according to standard of care. At visit 2, after randomization, assessments will be performed, after which subjects will initiate antibiotic pre-treatment with vancomycin (4 x 1 oral capsule containing 125 mg of vancomycin hydrochloride daily) for 6 days, followed by a washout period of a minimum of 1 day (>24 hours) to a maximum of 3 days (<72 hours). Treatment will start at visit 3 (baseline), immediately after the antibiotic washout period. At visit 3 (baseline), the inclusion and exclusion criteria will, if not met, also qualify as discontinuation criteria. Subjects not meeting the relevant inclusion and exclusion criteria at visit 3 will be discontinued from the trial. Each subject will receive randomized blinded treatment once weekly for 8 weeks, and will be followed-up for 52 weeks after treatment initiation on day 1. In case a subject discontinues the trial, end of trial assessments should be performed as soon as possible (within 14 days) after a decision of trial discontinuation has been taken. A corticosteroid tapering protocol will be applied to assess corticosteroid-free clinical remission and response during the trial.

[0214] The dosing regimen is one EVA bag containing 150 mL RBX2660 or placebo per week, corresponding to one dose, for 8 weeks.Atty. Docket No. 052209-0664

[0215] Safety assessments will include recording of adverse events and concomitant medications, safety laboratory assessments (clinical chemistry, hematology, and coagulation), urinalysis, 12-lead electrocardiogram (ECG), assessment of vital signs (blood pressure and heart rate), and physical examination.

[0216] Starting at visit 2 (randomization), stool samples will be collected prior to each visit for analyses of fecal-associated microbiota (including taxonomic and functional profiling) and fecal calprotectin level. Fecal metabolomics will be analyzed at visit 2 (randomization; before antibiotic pre-treatment), visit 3 (baseline, before first IMP administration) and visits 4, 7, 11, 12, and 13 (weeks 1, 4, 8, 26, and 52; end-of-trial). Blood samples for analysis of serum fecal calprotectin level will be collected at all visits from visit 3 (baseline) throughout the trial, and blood samples for analysis of immune cell populations will be collected at visit 3 (baseline) and visits 4, 7, 11, 12, and 13 (weeks 1, 4, 8, 26, and 52; end of-trial).

[0217] Subjects will record stool frequency and rectal bleeding in an electronic daily diary. The diary is to be completed for 7 consecutive days just prior to visit 2 (randomization) and on a daily basis until visit 11 (week 8). The diary must also be completed for 7 consecutive days just prior to visits 12 and 13 (weeks 26 and 52; end of trial). The daily scores obtained from the subject electronic diary during the 7 days before the trial visit will be used to calculate Mayo subscores for stool frequency and rectal bleeding. Mayo subscores obtained at visit 2 (randomization) are considered baseline for all Mayo score-related assessments.

[0218] Colonoscopy will be performed at visit 2 (randomization) for calculation of baseline Mayo endoscopic subscore and confirmation of eligibility based on the modified Mayo score (consisting of the stool frequency, rectal bleeding, and endoscopic Mayo subscores). Colonoscopy will then be performed at visits 11 and 13 (weeks 8 and 52; end-of-trial) for assessment of endoscopic remission and endoscopic response, as well as clinical remission and clinical response based on the modified Mayo score. In addition, corticosteroid-free clinical and endoscopic remission and response will be assessed at visits 11 (week 8) and 13 (week 52; end of trial). The colonoscopy will be video recorded and transferred to independent central readers for endoscopic scoring.Atty. Docket No. 052209-0664

[0219] Physician’s global assessment will be performed at visits 2 (randomization), 11, 12, and 13 (weeks 8, 26, and 52; end-of-trial) for evaluation of clinical remission and response at visit 12 (week 26) based on the partial Mayo score (consisting of the Mayo subscores stool frequency, rectal bleeding, and physician’s global assessment), and allow for calculation of total Mayo score (consisting of all four Mayo subscores) throughout the trial.

[0220] Biopsies for mucosal microbiota analyses and assessment of histological disease activity (using NHI) will be taken as part of the colonoscopy procedure at visits 2 (randomization), 11 (week 8), and 13 (week 52; end-of-trial).

[0221] Changes from baseline in the use of biologies and / or small molecules for treatment of UC will be assessed at visits 11, 12, and 13 (weeks 8, 26, and 52; end-of-trial).Inclusion Criteria

[0222] Subjects must meet all of the criteria listed below to be eligible for participation:1. Signed informed consent obtained before any trial-related procedures at screening.2. Subject aged >18 at screening.3. Established diagnosis of ulcerative colitis (UC) by standard clinical, endoscopic and histological criteria, for more than 3 months at screening.4. Active mild to moderate UC as defined by a modified Mayo score >4 and <7, including an endoscopic subscore >2 and a rectal bleeding subscore >1 at randomization.5. Currently not treated, or on stable (i.e., no change in therapy and / or dose) treatment with 5-aminoasylic acid (5-ASA), immunomodulators (e.g., azathioprine (AZA), 6- mercaptopurine (6-MP), methotrexate, and cyclosporine), and / or corticosteroids to manage active UC: a) for at least 4 weeks prior to screening, and b) at randomization, and c) at baseline (visit 3).6. Treatment naive, or on stable (i.e., no change in therapy and / or dose) treatment with biologies and / or small molecules for treatment of UC: a) for at least 8 weeks prior to screening, and b) at randomization, and c) at baseline (visit 3).7. Adherence to at least one of the following conditions throughout the trial period: The subject, if female, must: a) be post-menopausal (women >45 years with no menstrual period for >12 months without an alternative medical cause), orb) have been surgicallyAtty. Docket No. 052209-0664 sterilized, or c) have had a hysterectomy prior to screening, or d) use an adequate method of contraception (i.e., implants, injectables, hormonal intrauterine devices, combined hormonal contraceptives, having a vasectomized sexual partner, or total abstinence from heterosexual relations with no plans of becoming pregnant through insemination or in vitro fertilization).The subject, if male, must: a) use a non-hormonal single-barrier contraception (i.e., condom), and b) use an adequate method of contraception if his female partner is of childbearing potential (i.e., not post-menopausal) as defined above. This is however not required if the male subject is documented surgically sterile, remains sexually abstinent, does not engage in heterosexual relations, or if he has a female partner who is surgically sterilized, had a hysterectomy, or is post-menopausal.8. Willing to abstain from consuming probiotics (including over the counter and prescription) from screening until 2 weeks after the last dose of investigational medicinal product (IMP).9. Willing to comply with trial procedures, including attending scheduled visits, completion of the electronic subject diaries, and adherence to treatment plan.Exclusion Criteria

[0223] Subjects meeting any of the criteria listed below will not be eligible for participation:1 . Active di sease or hi story of Crohn ’ s di sease.2. Active disease or history of irritable bowel syndrome (IBS) or any other chronic intestinal disease apart from UC.3. Active gastrointestinal infection.4. Major gastrointestinal tract surgery (this does not include appendectomy or cholecystectomy): a) within 60 days before screening, or b) between screening and randomization, or c) between randomization and baseline (visit 3).5. Planned major gastrointestinal tract surgery, e.g., colectomy.6. Previous failure, or loss of response, to 3 or more biologies and / or small molecules for treatment of UC.7. Use of >25 mg of prednisolone or equivalent corticosteroid dose per day.Atty. Docket No. 052209-06648. Use or planned use of systemic antibiotics for an indication other than the qualifying UC.9. Fecal microbiota transplantation (FMT), or treatment with other microbiota based therapies: a) within the past 6 months before screening, or b) between screening and randomization, or c) between randomization and baseline (visit 3).10. Compromised immune system including, but not limited to, inherited / primary and acquired immune disorders as judged by the investigator.11. Solid-organ transplant recipient.12. Initiation or escalation of systemic immunosuppressive agents, at the discretion of the investigator, for any condition: a) during the 8 weeks before screening, or b) between screening and randomization, or c) between randomization and baseline (visit 3).Subjects on a stable dose of systemic immunosuppressants (e.g., <20 mg / day prednisolone equivalent daily dose) may be treated at the discretion of the investigator.13. Clinically relevant comorbidities, including but not limited to cardiac, liver or renal failure, as judged by the investigator at screening.14. Current abuse of alcohol or drugs, as judged by the investigator at screening.15. Pregnant, as confirmed by a positive pregnancy test, breastfeeding, or intending to become pregnant, during trial participation.16. Known or suspected hypersensitivity to polyethylene glycol 3350.17. Severe food allergy, characterized as previous episodes of severe allergic reactions in the form of anaphylaxis after intake of food allergens.18. Known or suspected hypersensitivity to vancomycin.19. Life expectancy of <12 months, as judged by the investigator at screening.20. Participation in any interventional clinical trial during the past 12 weeks before screening.21. Inability to participate in the trial for other reasons, as judged by the investigator at screening.Example 6: Administration of RBX2660 By Colonoscopy to Adults with Cancer

[0224] Treatment with a MRT composition prior to surgical breast cancer treatment may significantly increase cytotoxic T cells and / or reduce regulatory T cells in both peripheral bloodAtty. Docket No. 052209-0664 and tumors. The increase in cytotoxic T cells and / or reduction of regulatory T cells may enhance the efficacy of the cancer treatment. In some of these and in other instances, treatment with a MRT composition may modulate the metabolic function of regulatory T cells.

[0225] The MRT composition used in this study may be any composition as disclosed herein, such as RBX2660, that is administered by colonoscopy in a manner similar to what is disclosed in Example 2.

[0226] Patients with breast cancer will be grouped into cohorts based on their breast cancer profile. Cohort 1 will include patients that are estrogen receptor (ER) and / or progesterone receptor (PR) positive only (e.g. no human epidermal growth factor receptor 2 (HER2) amplification or overexpression). Cohort 2 will include patients that test positive for human epidermal growth factor receptor 2 (HER2) along with ER and / or PR. Cohort 3 will include patients that are negative for ER, PR, and HER2.

[0227] Patients from each cohort will be administered a dose of the MRT composition by colonoscopy. Dosing regimen one will include administering the MRT composition by colonoscopy 2 weeks prior to surgery. Dosing regimen two will include administering the MRT composition 4 weeks prior to surgery. RBX2660 will be administered to the right side of the colon (i.e., between the ileocecal valve and the hepatic flexure of the colon).

[0228] Treatment with the MRT composition by colonoscopy may enhance the efficacy of the cancer treatment (e.g., surgical treatment of breast cancer).Example 7: Reduction of Antimicrobial Resistance Genes in Patients with rCDI Following RBX2660 Treatment

[0229] A comprehensive post-hoc analysis of antibiotic resistance genes (ARGs) in subjects enrolled in the phase 3 , placebo-controlled PUNCH CD3 clinical study (NCT03244644) was conducted to explore if treatment with the MRT composition RBX2660 (FDA-approved for administration via enema as REBYOTA®, also referred to as RBL) may help decolonize patients that carry antibiotic resistance genes (ARGs), e.g., in their gut microbiota. Clinical outcomes of the PUNCH CD3 study were previously reported. See Khanna, et al., Drugs 2022; 82: 1527-1538Atty. Docket No. 052209-0664(reporting a CDI treatment success rate of 70.6% for RBX2660 administered by enema versus 57.5% for placebo, with an estimated treatment effect of 13.1%).

[0230] The PUNCH CD3 trial was a prospective, multicenter, double-blind, placebo-controlled phase 3 trial assessing the efficacy and safety of a MRT composition as described herein (RBX2660; FDA-approved for administration via enema as REBYOTA®; also referred to as RBL) in participants with rCDI. Participants enrolled in the PUNCH CD3 trial were aged >18 years with documented rCDI, who received antibiotic treatment for CDI and, following a washout period of 24 to 72 hours, a single dose of rectally administered MRT composition (RBX2660, 150 mb microbiota suspension) or placebo (normal saline). (FIG. 4) Treatment success was defined as the absence of CDI diarrhea within 8 weeks of study treatment.

[0231] Participants’ stool samples, collected at baseline and 1 week, 4 weeks, 8 weeks, 3 months, and 6 months after treatment, were subjected to deep shotgun sequencing analysis (2 x 150 bp, ~20 M reads / sample). ARGs were characterized using the AMR++ v3.0 bioinformatic pipeline (Burrows-Wh eel er- Aligner alignment with SNP variant calling; hit filtering of 80% gene fraction) and MEGARes v3.0 database (acyclical annotation for count-based, hierarchical statistical analysis; CARD, BacMet, ResFinder, PointFinder, AMRFinder Plus). ARG richness, defined as the number of unique ARGs in a given sample, was assessed at the class (4 antimicrobial compound types, 59 resistance classes), mechanism (233), and group (1448) levels in MRT (“RBL”) and placebo-treated participants. A generalized mixed linear model was used to compare ARGs across timepoints and treatment outcomes. Overall ARG richness decreased significantly from before to after treatment at the class, mechanism, and group levels, and to a significantly greater extent in MRT -treated vs. placebo-treated subjects,

[0232] At baseline, the microbiomes of treatment responders differed from those of healthy populations, with gammaproteobacteria and bacilli being predominant at baseline whereas bacteroidia and clostridia are typically predominant in healthy populations As illustrated in FIG. 5, MRT treatment responders (RBL) showed increased bacteroidia by 1 week after treatment, and the microbiome shifts were sustained through at least 6 months after treatment.

[0233] FIG. 6A illustrates the multiple ARG classes detected at baseline for study participants (RBL and placebo) and healthy subjects. A median of 34 (interquartile range, 32 -35) ARG classesAtty. Docket No. 052209-0664 were identified per participant at baseline, which is significantly more ARG classes than in a representative healthy populations (median, 10, interquartile range, 7 - 15), and there was no significant difference between all study participants at baseline (p > 0.05). Also, no significant difference in the number of ARG classes among key population subgroups were observed (p > 0.05), including participants pre-treated with vancomycin vs. other antibiotics, participants with < 3 prior CDI episodes vs. those with > 3, and participants aged < 65 years old vs. those aged > 65. FIG. 6B illustrates clinically important ARG classes detected at baseline, which included betalactam ARGs (Beta.), tetracycline ARGs (Tetra.), glycopeptide ARGs (Glyco.), and fluoroquinolone ARGs (Fluro.).

[0234] FIG. 7 illustrates reduction in ARG class richness after MRT treatment (RBL) and placebo treatment. As early as 1 week after treatment, ARG class richness was significantly reduced compared with baseline (P < 0.05), and continued to decrease through 6 months. ARG class richness decreased to a significantly greater extent at all post-treatment timepoints in participants who received the MRT composition (RBL) versus placebo treatment (P < 0.05).

[0235] FIG. 8 illustrates changes in ARG richness for clinically important ARG classes, which included beta-lactam ARGs, tetracycline ARGs, glycopeptide ARGs, and fluoroquinolone ARGs, after MRT treatment (RBL) and placebo treatment. As early as 1 week after treatment, beta-lactam and fluoroquinolone ARG richness decreased compared with baseline, and continued to decrease through 6 months among all participants. Beta-lactam ARG richness decreased more after MRT composition treatment than placebo. Glycopeptide ARG richness was decreased at 3 and 6 months compared with baseline. Tetracycline ARG richness increased among all participants (tetracycline resistance is prevalent in commensal bacteria in healthy populations).

[0236] FIGs. 9A and 9B illustrate changes in ARG richness at the mechanism and group levels respectively, which decreased significantly after MRT composition treatment (RBL) and placebo treatment. Specifically, ARG richness at the mechanism and group levels decreased significantly from before to after treatment (P < 0.05). ARG richness at the mechanism and group levels decreased more after MRT composition treatment than placebo (P < 0.05).

[0237] FIG. 10 illustrates reductions in clinically critical ARG groups after MRT composition treatment (RBL). Specifically, most participants had >1 clinically critical ARG group at baselineAtty. Docket No. 052209-0664 and most decreased to <1 clinically critical ARG group after treatment. Clinically critical ARG groups included clinically critical gram-negative beta-lactam resistance ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF) and clinically critical colistin resistance ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ). Only a few participants (<10%) had vancomycin-resistance ARG groups (VanA, VanHA, VanXA) at baseline, and culture-based testing confirmed few VRE-positive samples at baseline.

[0238] Collectively, the results showed that ARG richness decreased significantly from before to after treatment at the class, mechanism, and group levels, and to a significantly greater extent in MRT composition-treated vs. placebo-treated participants. Notably, at the class level, fluoroquinolone and beta-lactam ARG richness significantly decreased at all timepoints up to 6 months (P<0.05) and significantly differed between MRT composition and placebo treatment responders (P<0.05). In addition, several clinically relevant ARG groups were found to decrease after treatment, including, for example, extended-spectrum beta-lactamases. These data indicate that treatment with an MRT composition as described herein can be effective for AMR gut decolonization.Example 8: Microbiome and Quality of Life Shifts After RBX2660 Treatment of rCDI

[0239] As noted above, rCDI is associated with gut microbiome dysbiosis causing significant mortality and morbidity and resultant worsening health-related quality of life (HRQOL). RBX2660 was effective in preventing rCDI in the phase 3, randomized, placebo-controlled clinical study PUNCH CD3 (NCT03244644) discussed above. This example reports a categorical statistical analysis that queried whether Cdiff32 HRQOL scores were more likely to improve after RBX2660 than placebo, and a Dirichlet-multinominal recursive partitioning model assessed whether Cdiff32 HRQOL scores were linked to fecal microbiome or metabolome compositions. Key supportive secondary outcomes of PUNCH CD3 for this analysis included sustained clinical response, defined as treatment success of the presenting CDI recurrence at 8 weeks and no new CDI episodes through 6 months after completing a study treatment, and HRQOL as measured using the C. diff Health-related Quality-of-Life (Cdiff32) questionnaire at baseline, weeks 1, 4, and 8, and months 3 and 6 (see FIG. 11).Atty. Docket No. 052209-0664Microbiome Analysis

[0240] Individuals who agreed to participate in fecal sample collection received kits to collect whole stool samples at baseline, and 1, 4, and 8 weeks, and 3 and 6 months after the blinded study treatment. Samples were sent for processing and were stored frozen at -80°C without the addition of any stabilizing agents until further analysis. Only fecal samples received at the requested timepoints were included for analysis; samples received after a treatment failure event were excluded from the analyses, since those participants may have received antibiotic and / or additional RBX2660 upon CDI recurrence. Fecal samples were extracted using a PowerSoil Pro kit (Qiagen, Germantown, MD) and subjected to shallow-shotgun sequencing (Diversigen, Minneapolis, MN), with sequencing reads processed to operational taxonomic unit (OTU) data. Relative taxonomic abundances were calculated using OTU data and per sample microbiome compositions were analyzed using a non-metric multidimensional scaling methodology.Bile Acid Analysis

[0241] Analysis of BAs was conducted as previously described in Papazyan, et al. Microorganisms 2023; 11 : 135. Briefly, wet fecal samples were suspended in saline to a concentration of 0.3 g / mL, prepared and stored at -50°C until analysis. A liquid chromatography tandem mass spectrometry method was used to quantify 31 fecal BAs based on standards. The total S:P ratio was calculated and evaluated as previously described.Categorical approach to evaluating change in HRQOL

[0242] Cdiff32 questionnaire responses measured at baseline and after RBX2660 or placebo administration were coded from highest to lowest HRQOL as follows: A > B > C > D > E, where the ‘>’ symbol indicates higher HRQOL to the left for all adjudicated treatment responders. For example, answers of Totally Disagree > Mostly Disagree > Don’t Know > Mostly Agree > Totally Agree to Question 25, “I feel depressed because of my C. diff infection,” were coded as A, B, C, D, or E, respectively.

[0243] To assess before to after administration changes, possible HRQOL response patterns for each question for each participant were categorized as shown in FIG. 12, with baseline responsesAtty. Docket No. 052209-0664 in rows and the last received response during the blinded phase in columns. Participants with higher HRQOL responses (A or B) at baseline were categorized as either Maintained (A or B) or Deteriorated (C, D, or E) at the last received response (FIG. 12). Conversely, participants with lower baseline HRQOL responses (C, D, or E) were categorized as either Improved (A or B) or Not Improved (C, D, or E) at the last received response. For most participants, the last response was at 8 weeks. For participants with incomplete diary entries at a given visit, first and last responses were tracked on a per question basis. For example, participant 1's first response to question 1 is at baseline (BL), while their first response to question 3 is at week 1 (1W).

[0244] Two statistical analyses of the possible response patterns were conducted for each Cdiff32 question to evaluate if RBX2660 impacted HRQOL more than placebo. The first compared Improved versus Not Improved as outcomes with study treatment entered as the predictor in a logistic regression model and odds ratios (ORs) were calculated. An OR >1 indicated RBX2660 responders were more likely to have Improved HRQOL versus placebo responders. The second analysis compared participants who Maintained HRQOL versus Deteriorated as an outcome, with study treatment entered as the predictor in a logistic regression model and the OR calculated. An OR >1 indicated RBX2660 responders were more likely to have Maintained HRQOL versus placebo responders.

[0245] In addition to analyses of individual Cdiff32 questions, the domain (mental, physical, social) and total Cdiff32 aggregate scores were analyzed. Logistic regression and generalized linear models to account for repeated measures were used with Improved / Not Improved or Maintained! Deteriorated as outcome and study treatment as predictor with OR >1 indicating HRQOL was Maintained or Improved to a greater extent after RBX2660 than placebo. ORs, 95% Cis, and ^-values were calculated for individual questions and for domain and total Cdiff32 scores. No adjustments were made for multiple testing.Microbiome recursive partitioning regression

[0246] To test if Cdiff32 mental domain HRQOL was associated with gut microbiome compositions, a regression analysis developed for microbiome taxa count data was used. See, Yang, et al., Sci. Rep. 2019; 9: 20082. Dirichlet-multinomial Recursive Partitioning (DM-RPart) regression combines recursive partitioning with the Dirichlet-multinomial probability distribution.Atty. Docket No. 052209-0664See, La Rosa, et al. PLoS One 2012; 7, e52078. This produced DM-RPart derived decision trees that partitioned microbiome samples into nodes or subgroups that exhibit more homogeneity based on similar input variable values. Partitions were discovered using the likelihood ratio from the Dirichlet-multinomial distribution. See, Yang, et al. The DM-RPart model was fit to data from all sample time points for which both Cdiff32 and microbiome data were available, with class-level taxonomic counts as the outcome or response variable and the Cdiff32 mental domain categorical HRQOL question responses coded from highest to lowest HRQOL (A > B > C > D > E) as the input or predictor variables. DM-RPart was fitted using 10-fold cross validation and minimum MSE (best tree).Bile Acid recursive partitioning regression

[0247] BAs from targeted metabolomics were measured as ng / g wet fecal weight. BA fractional abundance was calculated based on compositional data (fraction of total) and were partitioned using DM-RPart with HRQOL and other covariates as the input or independent variables. This model included all sample time points for which both Cdiff32 and BA data were available. Models were fitted with 10-fold cross-validation and trees selected with near minimum mean square error (MSE).Results - Change in HRQOL for Participants

[0248] Cdiff32 HRQOL questionnaire data from the study were analyzed categorically by coding each questionnaire response for each participant and time point from highest to lowest HRQOL as A > B > C > D > E, where A is highest and E lowest. At baseline, most responses were poorer (C, D, or E) for most questions and participants (see FIGs. 12 and 13), as is common among patients with rCDI. HRQOL changes after RBX2660 or placebo administration were assessed by categorizing the change from baseline to the last received response while still blinded (eight weeks for most participants) as either Improved from Baseline (A / B response at eight weeks) or Not Improved from Baseline (see FIG. 12). To focus on RBX2660 versus placebo differences rather than the influence of clinical outcome on HRQOL, this analysis only included participants adjudicated as clinical responders. Among the minority (38.2%) of participant responses that were better at baseline (A or B), post-treatment outcomes were categorized as Maintained from Baseline ox Deteriorated from Baseline (FIG. 12). Of the 267 participants in the ITT study population, 179Atty. Docket No. 052209-0664 clinical responders’ first and last blinded Cdiff32 questionnaire responses were available and included in the analysis.

[0249] Logistic regression was used to model Improved versus Not Improved from Baseline comparing RBX2660 vs placebo. The forest plot in FIG. 14 shows the odds ratios (ORs) and 95% confidence intervals for each HRQOL question and aggregate scores for each domain. ORs to the right of the vertical line at 1 indicate higher odds of improving in RBL compared with placebo responders. Participants with poorer responses at baseline had greater odds of improvement in HRQOL responses after RBX2660 than after placebo for all 14 mental domain questions (FIG. 14). The largest differences were questions about irritability or isolation. An additional regression that aggregated all mental domain questions and accounted for repeated measures also indicated overall mental domain HRQOL was more likely to be Improved after RBX2660 than placebo (OR: 2.04 [1.01; 4.12], / ><0.05). Most of the physical and social domain HRQOL questions and domain aggregates were numerically more likely to be Improved after RBX2660 than placebo (OR: 2.09 [0.88; 4.96] and 1.40 [0.14; 14.37] for physical and mental domain aggregates, respectively). An overall all-domain aggregate was also more likely to be Improved after RBX2660 than placebo (OR: 1.64 [0.91; 2.94]), which is consistent with responses to question 32 “How would you rate your overall quality of life?”.

[0250] Participants with better HRQOL responses at baseline (A or B) showed no difference in the odds of being Maintained after RBX2660 than placebo administration for some questions, and there was no significant clustering within a particular domain, and the all-domain aggregate likelihood of being Maintained was similar between RBL and placebo (See FIG. 15).Results - Differentiating microbiome compositions and associations with HRQOL

[0251] A prior exploratory analysis indicated PUNCH CD3 participants’ gut microbiome composition shifted after RBX2660 administration to have increased Clostridia and Bacteroidia and decreased Gammaproteobacteria and Bacilli, compared to baseline. To explore associations between these changes and HRQOL mental domain scores, sequencing read counts assigned at the taxonomic class level were regressed as dependent variables onto HRQOL responses (A, B, C, D, or E) for the 14 mental domain questions as input variables using a non-parametric regression model (Dirichlet-Multinomial Recursive Partitioning; DM-RPart). Any sample data for whichAtty. Docket No. 052209-0664 both microbiome and Cdiff32 data were available were included in this analysis, irrespective of study treatment received or clinical outcome, and spanning baseline to six months after administration.

[0252] The DM-RPart algorithm resulted in a tree in which the total data set (Node 1, FIG. 16, Panel A, N = 636) was split into eight terminal subgroups (Nodes 5 - 19). Each node / group was split into two subgroups (child nodes) based on responses to a single HRQOL question, with the two subgroups having more homogeneous microbiome compositions than the beginning (parent) node / subgroup (FIG. 16, Panel A). For example, the total data set (Node 1) was partitioned into two microbiome composition clusters based on responses to the question of “feeling isolated.” Those who answered ‘A’ (Node 2) had similar microbiome compositions that differed from the microbiome compositions among those who answered B through E (Node 11). The terminal subgroups (Nodes 5 through 19) were defined by sequential partitioning of multiple HRQOL questions. For example, Node 5 was defined by successive partitions of having more positive answers to the four questions related to: (i) not feeling isolated; (ii) feeling in control of CDI; (iii) feeling capable of living a normal life; and (iv) no fear of transmitting CDI to others. Although the DM-RPart algorithm fit a partition tree based on responses to only six HRQOL questions, the resulting terminal subgroups had a clear gradient of responses to all 14 mental domain questions. The microbiome composition of Node 5 had the highest relative abundance of Clostridia and the lowest relative abundance of Gammaproteobacteria among all terminal nodes (FIG. 16, Panel B, Table 5). Generally, the highest HRQOL responses were in Node 5 progressing left to right to lowest responses in Node 19 (FIG. 16, Panel C). Conversely, Node 19 — defined by lower Cdiff 2 HRQOL scores related to feelings of isolation and not being in control of CDI — had the lowest relative abundance of Clostridia and highest relative abundance of Gammaproteobacteria.Table 5. Relative Abundance with Standard Error of Bacteria According to Taxonomic Composition by Terminal NodesAtty. Docket No. 052209-0664

[0253] Within this gradient, Clostridia and Bacteroidia relative abundances were higher when HRQOL was higher, whereas Gammaproteobacteria, Bacilli, and Negativicutes were lower in these high-HRQOL nodes. Within Bacteroidia and Clostridia classes, Bacteroides, Blautia, Lachnosclostridium, Faecali bacterium, Roseburia, and Eubacterium were key genera that differed among terminal subgroups, whereas Klebsiella, Escherichia, Citrobacter, Lactobacillus, and Streptococcus SNQK, key genera the differed within the Gammaproteobacteria and Bacilli classes (FIG. 17, Tables 7-9). These data indicate a strong association between HRQOL responses and microbiome compositions, which further supports the microbiota-gut-brain axis (GBA) link, which is modulated through the CNS, immune and gastrointestinal functions.Atty. Docket No. 052209-0664Table 6. Relative Abundance of Gammaproteobacterial Genera by Terminal NodesTable 7. Relative Abundance of Clostridia Genera by Terminal NodesTable 8. Relative Abundance of Bacteroidia Genera by Terminal NodesTable 9. Relative Abundance of Bacilli Genera by Terminal NodesAtty. Docket No. 052209-0664Results - Differentiating BA compositions and associated HRQOL mental domain scores

[0254] Participants in PUNCH CD3 also shifted from primary bile acid (BA) predominance prior to study treatment to secondary BA predominance after administration, with larger shifts in RBX2660 versus placebo responders. To explore associations between these changes and Cdiff32 HRQOL scores, a DM-RPart analysis was conducted in which the concentration of 33 BAs (ng / mL) was regressed as the dependent variable against mental domain HRQOL scores as the input variables. Sample inclusion criteria were the same as for microbiome regression analyses. The partition tree with the lowest MSE had a single split into two subgroups, so the tree with the second lowest MSE was selected, slightly sacrificing overall accuracy for improved interpretability. This tree is within the MSE ± 1 SE rule commonly used in model selection (DM- RPart). The resulting tree split based on three HRQOL questions (FIG. 18, Panel A), with the resulting four terminal nodes having a gradient of higher to lower HRQOL responses among all mental domain questions. Among those four terminal nodes, secondary BA predominance was associated with higher HRQOL responses (secondary to primary BA ratio [S:P], Node 2, FIG. 18, Panel B) and primary BA predominance was associated with lower HRQOL responses (Nodes 12 and 15). This trend was also observed for the mean concentrations of several individual BAs, including higher lithocholic and deoxycholic acid (LCA, DCA) concentrations being associated with higher HRQOL responses and higher glycolic, cholic, and taurocholic acid (GCA, CA, TCA) concentrations being associated with lower HRQOL responses (FIG. 18, Panel C). The distribution of Cdiff32 HRQOL mental domain scores for the four terminal subgroups (Nodes 2 - 15) is shown in the bubble plot (FIG. 19), indicating the relative number of responses in each category (A - E). The relative size of the circles generally indicates that the highest HRQOL responses (larger circles) appear in Node 2 progressing left to right with the lowest responses (smaller circles) in Node 15.Discussion

[0255] In this study, the connection between gut dysbiosis and mental HRQOL was probed by leveraging the randomized, placebo-controlled PUNCH CD3 trial that included exploratory analyses of HRQOL as well as microbiota and metabolome changes. A categorical statistical approach was chosen because the Cdiff32 response data are categorical data (e.g., TotallyAtty. Docket No. 052209-0664Disagree, Mostly Disagree, etc.) and because it facilitated a highly intuitive comparison of the odds of participants reporting HRQOL improvements between RBL and placebo groups. By assessing only participants who were clinical responders (i.e., no recurrence), HRQOL differences related to study treatment administered, rather than clinical outcome, could be estimated. Responders were more likely to report Improved HRQOL after RBX2660 than placebo administration, particularly for mental domain questions. Mental domain questions related to anxiety — feeling irritable and / or isolated, fear of further diarrhea of antibiotics causing CDI again, or worry about transmitting CDI to family or friends — were the most improved in RBX2660 versus placebo responders. These results are consistent with numerical analyses that PUNCH CD3 participants experienced significantly greater increase in overall and mental domain Cdiff32 when administered RBX2660 compared to placebo.

[0256] The categorical analysis of Cdiff32 data enabled a regression analysis which indicated higher mental HRQOL is associated with higher relative abundance of Clostridia and Bacteroidia classes and lower relative abundance of Gammaproteobacteria and Bacilli. This is the first analysis to report concurrent and correlated longitudinal changes of mental HRQOL with microbiome changes, particularly within a controlled clinical trial of a microbiota-based product. The observed associations are consistent with cohort studies in which several Clostridia genera were associated with higher overall HRQOL or decreased depressive and anxiety symptoms. Clostridia and Bacteroidia species were also positively associated with better mental HRQOL in kidney transplant recipients. Therefore, this study increases the body of evidence that genera in these classes could be important drivers of mental HRQOL. A second regression analysis indicated higher mental HRQOL also correlated with higher ratios of secondary to primary BAs, as well as higher concentrations of LCA and DCA as key microbially derived secondary BAs. This is the first analysis of clinical trial data that correlated gut BA composition with mental HRQOL scores as measured by Cdiff32.

[0257] In conclusion, patient-reported HRQOL scores were comparable at partitioning microbiome composition as study treatment and outcome. After RBX2660 administration, higher mental HRQOL scores were associated with a gradient in increased Clostridia and Bacteroidia and decreased Gammaproteobacteria, Bacilli, and Negativicutes, which correlated with a shift from primary to secondary BAs. These analyses postulate that the microbiota-gut-brain axis is involvedAtty. Docket No. 052209-0664 in modulating central and peripheral mechanisms that link gut eubiosis and CNS functions and improving outcomes for patients with rCDI following RBL administration.Example 9: Safety and Efficacy of RBX2660 in Reducing rCDI in Immunocompromised Participants

[0258] Immunocompromising conditions increase the risk for primary and rCDI in individuals that are frequently hospitalized and exposed to antibiotic treatment. This example reports a subgroup analysis of the PUNCH CD3-OLS clinical trial (NCT03931941) that evaluated the safety and efficacy of MRT composition RBX2660 (FDA-approved for administration via enema as REB YOTA®, also referred to as RBL), for the subgroup of participants with rCDI and an immunocompromising condition and / or who were receiving immunosuppressive medication.Trial Design

[0259] The clinical trial was a phase 3, open-label, prospective, single arm study designed to evaluate the safety and tolerability of RBX2660 (as referred to as RBL) in participants with rCDI. Participants received a single 150 mb dose of RBX2660 administered rectally 24 to 72 hours after completing standard of care (SOC) antibiotics. Following a baseline assessment, data were collected using participant diaries (week 1), in office follow-up visits (week 1 and 8), and telephone assessments (week 4, months 4 and 6). Participants were eligible to receive a second course of treatment, with a single dose of RBX2660 within 21 calendar days of confirmed failure, if they met the criteria for treatment failure. The use of antibiotics prior to a second course of treatment was at the discretion of the investigator. If antibiotics were given to control symptoms, a 24 to 72 hour washout period was required prior to administration of the second course. For participants who received a second course of treatment, telephone assessments were conducted at 1 week, 8 weeks, and 6 months after administration of the second course of treatment.Study population

[0260] Clinical trial participants were aged >18 years with medical record documentation of rCDI, including first recurrence, or at least two episodes of severe CDI resulting in hospitalization. Participants had to be on a course of SOC antibiotics for CDI diarrhea at the time of enrollment and their diarrhea had to be controlled (defined as fewer than three unformed / loose stools / dayAtty. Docket No. 052209-0664[Bristol Stool Scale type 6—7] for two consecutive days prior to the antibiotic washout period). Notable exclusion criteria included: CD4 count <200 cells / mm3and / or absolute neutrophil count (ANC) of <1000 cells / pL at screening; receipt of FMT within the past 6 months; treatment with bezlotoxumab within the last year; and systemic antibiotic therapy for a condition other than CDI

[0261] This subgroup analysis included participants who were considered immunocompromised based on medical history at the time of RBX2660 administration and / or use of immunosuppressive medications for at least 2 weeks before RBX2660 administration to 8 weeks after RBX2660 administration. Medical conditions associated with impaired immune function were identified based on (1) Centers for Disease Control (CDC) guidelines for immunocompromised conditions (listed under CDC pneumococcal vaccination: end-stage renal disease, renal failure, asplenia, human immunodeficiency virus (HIV) or HIV infection, congenital hemoglobinopathies and immunodeficiency syndromes); and (2) medical conditions according to standardized Medical Dictionary for Regulatory Activities (MedDRA) queries. Also included were participants with end-stage liver disease. Other medical conditions of interest that were included based on indication for qualifying immunosuppressive medication (e.g., history of solid organ transplantation). Immunocompromising medications (e.g., immunosuppressive medications) were identified based on Anatomical Therapeutic Chemical (ATC) classification. Corticosteroids (“glucocorticoids” or “corticosteroids for systemic use”) were included if taken for 5 consecutive days within the prespecified medication use window. A dose of >20 mg prednisone / day or equivalent was considered to be an immunocompromising / immunosuppressive dose. Prednisone equivalent doses are listed in the below table. Non-corticosteroid immunocompromising / immunosuppressing medications included those with an ATC classification of “antineoplastic agents”, “immunostimulants”, or “immunosuppressants”. Only systemic administration routes were included (oral, intramuscular, subcutaneous, or intravenous injection).Table 10. Prednisone Equivalent Corticosteroid DosesAtty. Docket No. 052209-0664aprednisone equivalent doses are calculated by multiplying the dose of the medication by the conversion factor listed in the table (e.g., 4 mg dexamethasone is equivalent to 26.8 mg prednisone).

[0262] RBX2660 (described above) was provided as a pre-packaged 150 mL microbiota suspension and was administered rectally. Each dose contained between 1 * 108and 5* IO10colony forming units (CFU) / mL of fecal microbes including Bacteroides.Study outcomes

[0263] The primary endpoint for the clinical trial was the number of participants with RBX2660- or administration-related treatment-emergent adverse events (TEAEs) in the safety population. In this subgroup analysis, participants were classified as being immunocompromised or not immunocompromised for the safety analysis. TEAEs were defined as any adverse event (AE) occurring on or after RBX2660 administration. TEAEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA), version 20.0. Safety data were censored at the time of on study CDI recurrence (i.e., treatment failure). The severity of TEAEs, relatedness of TEAEs, and the number of select CDI-related TEAEs through 8 weeks after RBL administration (death, septic shock, toxic megacolon, colonic perforation, emergency colectomy, and intensive care unit admission) were assessed. Infection monitoring was included as part of the comprehensive TEAE assessment.

[0264] Efficacy outcomes included treatment success and sustained clinical response. Treatment success was defined as the absence of CDI diarrhea through 8 weeks of treatment. Sustained clinical response was defined as treatment success of the presenting rCDI and no new episodes of CDI through 6 months of completing treatment. Treatment failure (CDI recurrence) was defined as the presence of CDI diarrhea within 8 weeks of RBX2660 administration. Recurrence was confirmed by a positive stool test (C. DIFF QUIK CHEK COMPLETE® Test) for C. difficile glutamate dehydrogenase antigen and toxins A and B as determined by a central laboratory. If results were discordant (e.g., GDH-positive and toxin-negative), polymerase chainAtty. Docket No. 052209-0664 reaction (PCR)-based testing was performed. In this subgroup analysis, efficacy outcomes were also evaluated with consideration for select potentially clinically relevant variables (e g., number of immunocompromising medications).Results

[0265] Overall, 793 participants were enrolled in the trial and 697 were administered RBX2660. Of participants who were administered RBX2660, 141 participants (20.2%) were included in the immunocompromised (IC) subgroup. Eighty two (58.2%) participants were immunocompromised due to medications only, 41 (29.1%) participants had immunocompromising conditions only, and 18 (12.8%) participants were immunocompromised due to both immunocompromising medications and conditions. Most participants taking immunocompromising medications were taking one medication (n=61 (61.0%)), 23 (23.0%) were taking two medications, and 16 (16.0%) were taking three or more medications. According to the ATC classification, participants were taking systemic immunosuppressive medications, e.g., tumor necrosis factor alpha (TNF-a) inhibitors, interleukin inhibitors, and purine analogues (Table 11). The most frequent indications for immunosuppressive medications were IBD (n=42), arthritis (n=l 8), solid organ transplantation (n=12) and cancer (n=12). No participants had end-stage liver disease.Table 11. Summary of Immunocompromising Medications and ConditionsAtty. Docket No. 052209-0664aParti cipants could have >1 immunocompromising condition and be receiving multiple medications.bVedolizumab (n = 14), leflunomide (n = 7), mycophenolate mofetil (n = 6), my cophenolate sodium (n = 2), sirolimus, tofacitinib, tofacitinib citrate and upadacitinib (n = 1, one each).Atty. Docket No. 052209-0664cHydroxychloroquine (n = 9), methotrexate (n = 8), azathioprine (n = 6), hydroxychloroquine sulfate (n = 4), methotrexate sodium (n = 2) and lenalidomide (n =1).dUlcerative colitis (n = 26), Crohn’s disease (n = 14) and not otherwise specified (n = 2). ^Rheumatoid arthritis (n = 15), Psoriatic (n = 1) and not otherwise specified (n = 2).fKidney (n = 4), lung (n = 3), liver (n = 3) and not otherwise specified (n = 2).gAdenocarcinoma (n = 2), leukemia (n = 2), lymphoma (n = 2), Ewing’s sarcoma (n = 2) and others (n = 1).hSystemic Lupus Erythematosus, confirmed or suspected.

[0266] Baseline characteristics were comparable between participants in the IC and non-IC subgroups. Most participants were female (58.9% vs 72.7%) and white (89.4% vs 95.0%), and approximately half (48.2% vs 48.6%) were 65 years or older (Table 12). Most participants had three or fewer previous CDI episodes (IC subgroup, 69.5%; non-IC subgroup, 64.7%) and most participants in the IC (90.8%; 128 / 141) and non-IC (83.5%; 464 / 556) subgroups had received vancomycin for the enrolling CDI episode. The median SOC antibiotic course duration was 20.0 days (interquartile range [IQR] : 12.0-41.0 days) for participants in the IC subgroup, and 20.0 days (IQR: 13.0-35.0 days) for participants in the non-IC subgroup.Table 12. Participant Demographics and Baseline CharacteristicsAtty. Docket No. 052209-0664EIA: enzyme immunoassay; GDH: glutamate dehydrogenase; PCR: polymerase chain reaction;.“Enrolling tests could be used in combination and are not mutually exclusive. CDI diagnosis was determined by the treating physician; a positive test was not protocol specified.Atty. Docket No. 052209-0664bIncluded a loop-mediated isothermal amplification (LAMP) positive test result.Efficacy

[0267] In the modified intent to treat (mITT) population (N=676), treatment success (defined as the absence of CDI diarrhea through 8 weeks post-treatment) was achieved in 75.7% (106 / 140) of participants in the IC subgroup and 73.3% (393 / 536) of participants in the non-IC subgroup (Table 13). In participants with treatment success, sustained clinical response through 6 months was maintained in 88.7% (94 / 106) and 91.6% (360 / 393) of those in the IC and non-IC subgroups, respectively. Efficacy outcomes were comparable across select further subgroups: by number of previous CDI episodes; CDI antibiotic duration; number of immunocompromising medications; and type of immunocompromising medications (Table 14). In the tables that follow, only participants with adjudicated treatment outcomes are included. Efficacy outcome was deemed “indeterminate” if CDI test was inconclusive or missed at time of visit.Table 13. Treatment Success Within 8 Weeks and Sustained Clinical Response Within 6 Months (mITT Population)Table 14. Summary of Treatment Outcomes by Select Subgroups Treatment Success Within 8 Weeks (mITT population)Atty. Docket No. 052209-0664Cort: corticosteroid; Non-Cort: non-corticosteroid.Table 15. Summary of Treatment Outcomes by Select Subgroups - Sustained Clinical Response at 6 Months in 8-Week Responders (mITT Population)Cort: corticosteroid; Non-Cort: non-corticosteroid.Atty. Docket No. 052209-0664Second Course of Treatment

[0268] Of the 31 and 120 participants who met the criteria for treatment failure in the IC and non-IC subgroups, respectively, 23 in the IC subgroup and 98 in the non-IC subgroup opted to receive a second course of treatment within 21 days of treatment failure. In sum, 16.3% (23 / 141) of participants in the IC subgroup and 17.6% (98 / 556) of participants in the non-IC subgroup received two courses of single-dose RBX2660. The incidence of TEAEs following a second course of RBL were comparable between the IC and non-IC subgroups and were similar to those following a single course of RBL (Table 16). No new safety signals were observed.Table 16. Summary of TEAEs Within 8 Weeks and Between 8 Weeks and 6 Months of Second Course of RBX2660 (Safety Population)Atty. Docket No. 052209-0664aRelatedness categories are not mutually exclusive.

[0269] Following a second course of treatment, 39.1% participants in the IC subgroup and 59.2% participants in the non-IC subgroup achieved treatment success (Table 17). Of the participants who achieved treatment success following a second course, 88.9% and 87.9% of participants in the IC and non-IC subgroups, respectively, had a sustained clinical response at 6 months. Overall, 82.1% (115 / 140) of participants in the IC subgroup and 84.1% (451 / 536) of participants in the non-IC subgroup achieved treatment success, regardless of the number of administrations.Atty. Docket No. 052209-0664Table 17. Summary of Treatment Outcomes Following Second Course of RBX2660 (mITT population)aThe protocol-specified definition of treatment success or treatment failure was not met.bThe percentage of participants with sustained clinical response at 6 months is calculated with the number of participants with treatment success as the denominator.Discussion

[0270] This subgroup analysis is the first report of outcomes for the 141 immunocompromised participants that received RBX2660 in the PUNCH CD3-OLS clinical trial, including those with a history of solid organ transplantation, inflammatory bowel disease, arthritis, and cancer. None of the participants in the IC subgroup had a history of end-stage liver disease. This analysis provides important data regarding the safety and efficacy of RBX2660 for rCDI in mildly to moderately immunocompromised patients, which can assist providers during the clinical decision making process in real world care settings.

[0271] The results of this study found the safety and efficacy of RBX2660 in immunocompromised patients to be comparable with the overall study population. Most participants in the IC and non-IC subgroups received one course of single-dose RBX2660, consistent with the FDA-approved dosing for REBYOTA®. Most TEAEs were mild to moderate in severity, gastrointestinal in nature, and assessed as related to preexisting conditions or CDI. Serious TEAEs were reported more frequently in IC compared to non-IC participants, though no trends emerged to suggest a safety signal. In this study, no infections in the IC subgroup were considered to be related to RBL or its administration per both the treating clinician, study medicalAtty. Docket No. 052209-0664 monitor, and study DSMB. There were no reported instances of bacteremia or fungemia in these higher risk participants. No additional TEAEs were observed with two versus one course of singledose RBL. RBL appears to be safe in a mild-moderately immunocompromised patient population. Efficacy outcomes were comparable in the IC and non-IC subgroups, and consistent with those reported for the overall PUNCH CD3-OLS population and participants in the PUNCH CD3 trial, which excluded participants with immunocompromising conditions. Additionally, efficacy outcomes were comparable across select subgroups, including based on the number of previous CDI episodes, CDI antibiotic duration and both the number and type of immunocompromising medications.

[0272] The results of this subgroup analysis suggest RBX2660 can be used safely and is efficacious in immunocompromised patients.

Claims

1. Atty. Docket No. 052209-0664WHAT IS CLAIMED IS:

1. A method for treating recurrent Clostri di aides difficile infection (CD I) in a human patient in need thereof, comprising: administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

2. A method for treating a gastrointestinal disease or disorder in a human patient in need thereof, comprising: administering a dose of a microbiota restoration therapy composition to the patient rectally or by colonoscopy, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

3. The method of claim 2, wherein the gastrointestinal disease or disorder is one or more selected from ulcerative colitis and Crohn’s disease, optionally wherein the gastrointestinal disease or disorder is mild to moderate ulcerative colitis.

4. The method of claim 2, wherein the gastrointestinal disease or disorder is one or more selected from irritable bowel syndrome, spastic colon, functional bowel disease (FBD), non-ulcer dyspepsia (NUD), gastroesophageal reflux, inflammatory bowel disease, idiopathic or simple constipation, diverticular disease, AIDS enteropathy, small bowel bacterial overgrowth, celiac / coeliac disease, polyposis coil, colonic polyps, chronic idiopathic pseudo obstructive syndrome, chronic gut infections with specific pathogens, and viral gastrointestinal disorders.Atty. Docket No. 052209-06645. A method for treating a human patient suffering from cancer, comprising: administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

6. A method for reducing antimicrobial resistance gut colonization in a human patient in need thereof, comprising: administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

7. A method for altering gut microbiota composition in a human patient in need thereof, comprising: administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.

8. A method for altering gut metabolome composition in a human patient in need thereof, comprising: administering a dose of a microbiota restoration therapy composition to the patient by colonoscopy or rectally, wherein the microbiota restoration therapy composition comprises a fecal microbiota suspension comprising: fecal microbes derived from a human fecal sample, and a solution of polyethylene glycol and saline.Atty. Docket No. 052209-06649. The method of any one of the preceding claims, wherein the dose of the composition comprises from about 1 X 108to about 5 X IO10colony-forming units (CFU) / mL of fecal microbes.

10. The method of any one of the preceding claims, wherein the fecal microbes comprise spore-forming microbes and non-spore forming microbes.

11. The method of any one of the preceding claims, wherein the fecal microbes comprises microbes of species Bacteroides.

12. The method of any one of the previous claims, wherein the dose of the composition comprises > 1x1 CP CFU / mL of Bacteroides.

13. The method of any one of the preceding claims, wherein the fecal microbes are obtained by a process comprising processing a donor human fecal sample with a solution comprising polyethylene glycol and saline, and obtaining a filtrate comprising the fecal microbes.

14. The method of any one of the previous claims, wherein the solution comprises from 20 g to 50 g polyethylene glycol per 1 L of saline.

15. The method of any one of the previous claims, wherein the solution comprises 30 g polyethylene glycol per 1 L of saline.

16. The method of any one of the previous claims, wherein the polyethylene glycol comprises polyethylene glycol 3350.

17. The method of any one of the previous claims, wherein the saline is a 0.9% solution of sodium chloride.

18. The method of any one of the previous claims, wherein the dose of the composition has a volume of about 150 mL.

19. The method of any one of the previous claims, wherein the dose of the composition comprises no more than about 6 grams of polyethylene glycol.Atty. Docket No. 052209-066420. The method of any one of the previous claims, wherein the dose of the composition comprises no more than 5.97 grams of polyethylene glycol in saline.

21. The method of any one of the previous claims, wherein the dose of the composition comprises from about 150 g to about 192 g of the solution of polyethylene glycol in saline.

22. The method of any one of the previous claims, wherein the dose of the composition has a microbial colony forming unit (CFU) count of at least about 10 CFU per plate at a serial dilution of 104when cultured on a Bacteroides Bile Esculin Agar (BBE) culture plate.

23. The method of any one of the previous claims, wherein the dose of the composition has a microbial colony forming unit (CFU) count of from about 2.0 x 107CFU per mL to about 5.0 x 1010CFU per mL when cultured on a Center for Disease Control (CDC) culture plate.

24. The method of any one of the previous claims, wherein the dose of composition has bacteria from at least three colony types when cultured on a Center for Disease Control (CDC) culture plate.

25. The method of any one of the previous claims, wherein the composition includes bacteria from at least seven different families.

26. The method of any one of the previous claims, wherein the composition has a Shannon Diversity Index of 0.4-2.5 when calculated at the family level.

27. The method of any one of the previous claims, wherein the composition is administered by colonoscopy to the right side of the colon.

28. The method of any one of the previous claims, wherein the composition is administered by colonoscopy between the ileocecal valve and the hepatic flexure of the right side of the colon.

29. The method of claim 1, wherein the composition is administered by colonoscopy to the right side of the colon, between the ileocecal valve and the hepatic flexure of the right side of the colonAtty. Docket No. 052209-066430. The method of any one of the previous claims, wherein prior to treatment with the microbiota restoration therapy composition the patient has had one or more episodes of a recurrent CDI.

31. The method of any one of the previous claims, wherein prior to treatment with the microbiota restoration therapy composition the patient has been identified as having had one or more episodes of a recurrent CDI.

32. The method of any one of the previous claims, wherein prior to treatment with the microbiota restoration therapy composition the patient has been identified as having had a stool test positive for C. difficile or C. difficile toxin associated with the CDI.

33. The method of any one of the previous claims, wherein prior to treatment with the microbiota restoration therapy composition the patient has completed a course of antibiotic therapy for treatment of CDI.

34. The method of any one of the previous claims, wherein prior to treatment with the microbiota restoration therapy composition the patient has been identified as having completed at least one course of antibiotic therapy for treatment of CDI.

35. The method of claim 33 or claim 34, wherein the course of antibiotic therapy comprised at least 10 days of antibiotic therapy.

36. The method of any one of claims 33-35, wherein the microbiota restoration therapy composition is administered 24 to 72 hours after a last dose of the antibiotic therapy for CDI.

37. The method of any one of the previous claims, wherein prior to treatment with the microbiota restoration therapy composition the patient has been identified as not having experienced CDI diarrhea for two consecutive days.

38. The method of any one of the previous claims, wherein the patient does not experience CDI diarrhea for at least 8 weeks following treatment with the microbiota restoration therapy composition.Atty. Docket No. 052209-066439. The method of claim 1, wherein efficacy of the method is at least non-inferior to a method comprising administering the microbiota restoration therapy composition by enema.

40. The method of claim 1, wherein the method has a clinical efficacy rate of 75% or greater.

41. The method of claim 1, wherein the method has a clinical efficacy rate of 90 % or greater.

42. The method of claim 1, wherein the method has a clinical efficacy rate of 100%.

43. The method of claim 1 or claim 6, wherein the method reduces antibiotic resistance gene(ARG) richness in a sample obtained from the patient as compared to ARG richness prior to treatment, optionally wherein the method reduces richness of one or more clinically important ARG groups selected from gram-negative beta-lactam resistance-associated ARG groups, fluoroquinolone resistance-associated ARG groups, colistin resistance-associated ARG groups, and vancomycin-resistance-associated ARG groups.

44. The method of claim 43, wherein the method reduces one or more selected from betalactam ARG richness, fluoroquinolone ARG richness, glycopeptide ARG richness, and colistin ARG richness.

45. The method of claim 43, wherein the method reduces ARG richness of one or more clinically critical ARG groups selected from clinically critical gram-negative beta-lactam resistance ARGs (such as CTX, TEM, SHV, GES, BEL, OXY, KPC, IMI, IMP, and NDM), clinically critical fluoroquinolone resistance ARGs (such as GYRA, GYRB, GYRC, PARC, and PAREF) and clinically critical colistin resistance ARGs (such as MCR, LPXA, LPXC, PHOB, PHP, and PHOQ).

46. The method of claim 43, wherein the method increases tetracycline ARG richness.

47. The method of claim 1, wherein the patient is immunocompromised, optionally wherein the treatment is as effective in immunocompromised patients as in non-immunocompromised patients.Atty. Docket No. 052209-066448. The method of any one of the previous claims, wherein the patient is immunocompromised by having a one or more diseases or conditions selected from neoplasms, renal and urinary disorders, immune system disorders, HIV infection, and thalassemia at the time of administration of the microbiota restoration therapy composition.

49. The method of any one of the previous claims, wherein the patient is immunocompromised by having been treated with one or more immunocompromising medications for at least 2 weeks before administration of the microbiota restoration therapy composition.

50. The method of any one of claims 48 or 49, wherein the patient has one or more diseases or disorders selected from inflammatory bowel disease, arthritis, solid organ transplantation, cancer, systemic lupus erythematosus, multiple sclerosis, psoriasis, and scleroderma.

51. The method of claim 1, wherein the method results in improvement in quality of life as assessed by a C. t / / / / Heal th -Related Quality-of-Life (Cdiff32) questionnaire, optionally wherein the improvement relates to one or more of the physical, mental and social domains of the questionnaire, further optionally wherein the improvement relates to the mental domain of the questionnaire.

52. The method of any one of the previous claims, wherein the method results in improvement in quality of life relating to one or more of anxiety, coping with disease / health perception, control of disease, impact of stress, dysphoria, relationships, social reaction, daily activities, diet, sleep, and discomfort.

53. The method of claim 1 or claim 7, wherein the method increases relative abundance of one or more of Clostridia and Bacteroidia in the subject’s gut microbiome.

54. The method of claim 53, wherein the method increases relative abundance of one or more of Blautia, Lachnosclostridium, Faecalibacterium , Rose bur let, Eubacterium, and Bacteroides in the subject’s gut microbiome.

55. The method of claim 1 or claim 7, wherein the method decreases relative abundance of one or more of Gammaproteobacteria, Bacilli and Negativi cutes in the subject’s gut microbiome.Atty. Docket No. 052209-066456. The method of claim 55, wherein the method decreases relative abundance of one or more of Klebsiella, Escherichia, Citrobacter, Lactobacillus, and Streptococcus in the subject’s gut microbiome.

57. The method of claim 1 or claim 8, wherein the method increases a ratio of secondary bile acids to primary bile acids (S:P) in the subject’s gut metabolome.

58. The method of claim 1 or claim 8, wherein the method increases one or more of deoxycholic acid (DCA) and lithocholic acid (LCA) concentration in the subject’s gut metabolome.

59. The method of claim 1 or claim 8, wherein the method decreases one or more of cholic acid (CA), glycocholic acid (GA), and taurocholic acid (TCA) concentration in the subject’s gut metabolome.

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