GUT MICROBIOTA FOR THE TREATMENT OF HEART FAILURE WITH PRESERVED EJECTION FRACTION (HFpEF)

Abstract

The present invention relates to method and compositions of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, by administering to the subject a composition including gut bacterial populations and / or metabolites which are missing in the HFpEF subject compared to an individual not having HFpEF, or agents which reduce the levels of gut bacterial populations and / or metabolites which are present in the HFpEF subject in excess compared to a subject not having HFpEF.

Description

[0001] GUT MICROBIOTA FOR THE TREATMENT OF HEART FAILURE WITH PRESERVED EJECTION FRACTION (HFpEF)

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to treatment of cardiac disease. More specifically, the invention relates to treating heart failure with preserved ejection fraction (HFpEF) by transplanting a healthy microbiome or components thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Heart failure with preserved ejection fraction (HFpEF) represents a significant subset of heart failure cases, characterized by impaired cardiac function despite the preservation of left ventricular ejection fraction (LVEF). Current treatments for HFpEF aim to alleviate symptoms and manage comorbidities but do not effectively address the underlying pathophysiology, resulting in limited efficacy. This therapeutic challenge is exacerbated by the heterogeneous nature of HFpEF, with distinct phenotypes exhibiting varied clinical presentations and responses to treatment. Among these phenotypes, the obesity-related phenotype stands out due to its prevalence and association with metabolic disorders, including insulin resistance, type 2 diabetes, and hypertension. Obesity-driven systemic inflammation is a key contributor to HFpEF pathogenesis, implicating metabolic dysfunction as a critical determinant of disease severity. Importantly, emerging evidence suggests a bidirectional relationship between the gut microbiome and its metabolites and metabolic diseases, wherein dysbiosis, characterized by alterations in microbial composition and function, contributes to inflammation and exacerbates disease progression.

[0006] Accordingly, there is a need for developing gut microbiome-based therapeutics, especially directed to obesity-related phenotypes.

[0007] SUMMARY OF INVENTION

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0009] In some embodiments, there is provided a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, including administering to the subject at least one composition including: a) fecal material from a subject not diagnosed with HFpEF; b) a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c) at least one antibiotic agent capable of reducing the abundance or activity of gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d) at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and e) at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0010] In some embodiments, the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are anaerobic bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

[0011] In some embodiments, the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

[0012] In some embodiments, the metabolite is selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

[0013] In some embodiments, the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0014] In some embodiments, there is provided a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, including administering to the subject at least one composition including: a) fecal material from a subject not diagnosed with HFpEF; b) a therapeutically effective amount of isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae; c) at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus; d) at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy- D-proline, and phenylacetylglycine; and / or e) at least one agent that blocks production or activity of a small molecule metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0015] In some embodiments, there is provided a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, including administering to the subject at least one composition including: a) fecal material from a subject not diagnosed with HFpEF; and / or b) a therapeutically effective amount of isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0016] In some embodiments, the composition includes a therapeutically effective amount of at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0017] In some embodiments, the composition includes a therapeutically effective amount of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 bacterial families selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0018] In some embodiments, the composition includes a therapeutically effective amount of at least 2, at least 3, at least 4, at least 5, at least 6, or 7 bacterial genera selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0019] In some embodiments, the composition includes a therapeutically effective amount of at least one bacterial species selected from Parabacteroides species distasonis and Clostridium species aldenense.

[0020] In some embodiments, the subject in need of treatment is obese. In some embodiments, the subject in need of treatment is suffering from a disease or disorder selected form obesity, insulin resistance, type 2 diabetes, hypertension, and dysbiosis.

[0021] In some embodiments, the fecal material is a fresh fecal material.

[0022] In some embodiments, the fecal material has been cryopreserved.

[0023] In some embodiments, the fecal material includes at least one bacterial family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae. In some embodiments, the fecal material includes at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0024] In some embodiments, the isolated gut-associated bacteria are isolated from fecal material.

[0025] In some embodiments, the isolated gut-associated bacteria are cultured in vitro.

[0026] In some embodiments, the composition further includes saline and / or polyethylene glycol.

[0027] In some embodiments, the administration of the composition is a rectal administration.

[0028] In some embodiments, the administration of the composition is an oral administration.

[0029] In some embodiments, the isolated gut-associated bacteria are administered orally.

[0030] In some embodiments, the at least one composition is more than one composition.

[0031] In some embodiments, the method further includes a step of administering antibiotics prior to administering the composition. In some embodiments, the method further includes a step of administering antibiotics following administering the composition.

[0032] In some embodiments, there is provided a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, including administering to the subject at least one composition including: a) at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus; b) at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy- D-proline, and phenylacetylglycine; and / or c) at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0033] In some embodiments, the Lachnospiraceae genus is not Blautia or Clostridium. In some embodiments, the Lachnospiraceae genus is not Blautia. In some embodiments, the Lachnospiraceae genus is not Clostridium.

[0034] In some embodiments, the subject is obese. According to the current definition, an obese subject is a subject with a BMI (body mass index) of over 30.

[0035] In some embodiments, the composition includes at least 2, at least 3, or 4 metabolites selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0036] In some embodiments, the composition includes at least 2, at least 3, at least 4, or 5 agents that block production or activity of at least 2, at least 3, at least 4, or 5 metabolites selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0037] In some embodiments, there is provided a composition for use in a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject, the composition including at least one of: a) fecal material from a subject not diagnosed with HFpEF; b) a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c) at least one antibiotic agent capable of reducing the abundance or activity of gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d) at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and e) at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0038] In some embodiments, the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are anaerobic bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

[0039] In some embodiments, the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

[0040] In some embodiments, the metabolite is selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

[0041] In some embodiments, the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0042] In some embodiments, there is provided a composition for use in a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject, the composition including at least one of: a) fecal material from a subject not diagnosed with HFpEF; b) a therapeutically effective amount of isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae; c) at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus; d) at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy- D-proline, and phenylacetylglycine; and e) at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0043] In some embodiments, the Lachnospiraceae genus is not Blautia or Clostridium. In some embodiments, the Lachnospiraceae genus is not Blautia. In some embodiments, the Lachnospiraceae genus is not Clostridium.

[0044] In some embodiments, the composition includes a therapeutically effective amount of at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0045] In some embodiments, the composition includes a therapeutically effective amount of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 bacterial families selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0046] In some embodiments, the composition includes a therapeutically effective amount of at least 2, at least 3, at least 4, at least 5, at least 6, or 7 bacterial genera selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0047] In some embodiments, the composition includes a therapeutically effective amount of at least one bacterial species selected from Parabacteroides species distasonis and Clostridium species aldenense.

[0048] In some embodiments, the subject is obese. In some embodiments, the subject is suffering from a disease or disorder selected form obesity, insulin resistance, type 2 diabetes, hypertension, and dysbiosis.

[0049] In some embodiments, the fecal material is a fresh fecal material.

[0050] In some embodiments, the fecal material has been cryopreserved.

[0051] In some embodiments, the fecal material includes at least one bacterial family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0052] In some embodiments, the fecal material includes at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0053] In some embodiments, the isolated gut-associated bacteria are isolated from fecal material.

[0054] In some embodiments, the isolated gut-associated bacteria are cultured in vitro.

[0055] In some embodiments, the composition includes at least 2, at least 3, or 4 metabolites selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0056] In some embodiments, the composition includes at least 2, at least 3, at least 4, or 5 agents that block production or activity of at least 2, at least 3, at least 4, or 5 metabolites selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0057] In some embodiments, the composition further includes saline and / or polyethylene glycol.

[0058] In some embodiments, the composition is formulated for rectal administration.

[0059] In some embodiments, the composition is formulated for oral administration.

[0060] In some embodiments, the subject was treated by administering antibiotics prior to administering the composition. In some embodiments, the subject was treated by administering antibiotics following administering the composition.

[0061] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0062] BRIEF DESCRIPTION OF DRAWINGS

[0063] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0064] Figs. 1A-1D show characterization of the two-hit mouse model. Fig. 1A. A diagram representing the experimental setup for establishing the two-hit HFpEF mouse model. Fig. IB. Echo measurements of mice diastolic and systolic function, including e' Wave (graph at upper leftside), E / e’ ratio (graph at upper right-side), left ventricle global longitudinal strain (GLS) (graph at lower left-side), and LV Ejection fraction (LVEF) from m-mode (graph at lower right-side). Bars from left (each treatment group): CHOW (control), HFD (obese), HFpEF (HFD + L-NAME). Fig. 1C. Inflammatory fibrosis in the myocardium. The number of cells positive to the indicated staining was divided by the measured myocardial tissue area. A binary area is an area positive for both the indicated biomarker (CDl lb or CD31) and for the nuclear DAPI staining. Upper left panel: cells positive for CDl lb; upper right panel: cells positive to CD31; lower panel: a graph quantifying fibrotic areas in the myocardium based on tissue scarring identified with Masson’s tri chrome staining. The collagen-positive area was quantified as the proportion of collagen staining relative to the total tissue area using a custom MATLAB script. Bars from left: CHOW (control), HFD (obese), HFpEF (HFD + L-NAME). Fig. ID. A Principal Coordinates Analysis (PCoA) of the beta diversity of the gut microbiome, comparing samples from human groups (upper panel - controls: non-obese and non-HFpEF, obese-control: non-HFpEF, obese HFpEF: patients); and mouse groups (lower panel - CHOW: controls, L-NAME, HFD: obese, HFpEF: HFD + L- NAME), for visualizing the overall diversity and distance between the microbial communities across different samples.

[0065] Figs. 2A-2D describe humanized microbiota two-hit mice (7 weeks of feeding with HFD and L-NAME) following Fecal Microbiota Transplant (FMT). Fig. 2A. Schematic structure of the FMT experiment, showing the transfer process from humans to mice and subsequent tests conducted to assess the impact on gut microbiota and health outcomes. Fig. 2B. Graphs of diastolic and systolic parameters from echo for the mice groups. Upper left: e'Wave; upper right: E / e’ ratio; lower left: left ventricle (LV) global longitudinal strain (GLS); and lower right: LV Ejection fraction (EF) from m-mode. Bars (left to right): FMT from human control; FMT from human obese; FMT from human obese-HFpEF. Fig. 2C. Quantification of signal intensity in myocardium sections from mice strained with immunofluorescent antibodies against CD3, CD68, and CD31, identifying T cells, macrophages, and endothelial cells, respectively, upper left panel: CD3; upper right panel: CD68; lower panel: CD31. Fig. 2D. Measurement of cytokine levels in the plasma of the mouse groups. Upper left: fFNy; upper right IL-1P; lower left: IL-17; lower right: TNFa. It quantifies various inflammatory markers, illustrating the immune response dynamics following the FMT procedure. Left to right: FMT from human control; FMT from human obese; FMT from human obese-HFpEF.

[0066] Figs. 3A-3F present analyses of genes, bacterial populations and metabolites, related to HFpEF in FMT mice. Fig. 3A. The biological functions enrichment from Obese-HFpEF FMT mice vs. obese only, associated with Ingenuity Pathways Analysis (IP A) canonical positive Z- score: upregulated; negative Z-score: downregulated. Fig. 3B. Microbiome Sequencing, a Principal Coordinates Analysis (PCoA) graph representing the beta diversity among the microbial communities from different FMT mice groups. Fig. 3C. a Linear Discriminant Analysis Effect Size (LEfSe) graph displaying the key bacterial families that significantly explain the differences between the obese-HFpEF FMT and the obese only FMT groups. Bacterial families appearing more than once resulted from using operation taxonomy units (OTU) barcodes for generating the graph, and correspond to different versions of 16S rRNA which could not be unambiguously assigned to a species. Negative LDA score: under-represented in HFpEF FMT mice; Positive LDA score: over-represented in HFpEF FMT mice. Figs. 3D-3F. Metabolomics analyses, exploring the distinct metabolic fingerprints of systemic versus organ-specific responses, providing a detailed overview of the metabolic changes and interactions between obese-HFpEF and obese-only FMT mice groups. Fig. 3D. Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) derived from humanized microbiota mice plasma (upper panel) and heart tissue (lower panel). Component 1, Component 2, and Component 3 represent the first three dimensions extracted by the sPLS-DA. These components capture the parameters (dimensions) that best separate the experimental groups based on the variation in the data (e.g., metabolites) that contribute most to group discrimination. The percentage next to each component indicates how much of the total variance is explained by that component. Figs. 3E-3F. Pathway analysis of FMT obese-HFpEF vs. FMT obese only in humanized microbiota mice plasma (Fig. 3E) and heart tissue (Fig. 3F). Colors ranging from white to black indicate an increase in -p-value (loglO). The Y-axis (-loglO(p)) represents the statistical significance of each pathway, where higher values indicate lower p-values and therefore stronger evidence that the pathway is differentially regulated between the groups, while the size of the circle indicates an increase in "pathway impact" (weight of the metabolites relative change between the groups together with the importance of the metabolite that changed within the particular biological pathway). Therefore, the upper right quadrant becomes the most important, both in terms of statistical significance and biological significance.

[0067] Fig 4 presents an outline of the overall structure of the cross-FMT (therapeutics) experimental design. Generally, mice were treated with L-NAME only until week 7 and fed with HFD throughout the experiment. After week 7, mice were administered with an antibiotics cocktail (Abx: vancomycin hydrochloride, neomycin trisulfate salt hydrate, metronidazole, and sodium ampicillin) for 2 weeks, and a first FMT from obese-HFpEF donors, obese-only donors, or control donors was performed at week 9, to generate humanized mice. Mice were again administered with an antibiotics cocktail (Abx) for 2 weeks starting from week 13, and a second FMT was performed on the humanized mice groups at week 15, generating double FMT groups.

[0068] Figs. 5A-5G present analyses of the double FMT experiment. Fig. 5A. A graph displaying mice weight gain (%) across the double FMT experimental groups throughout the duration of the study. Week 7 is the end of the L-NAME treatment. Weeks 7-9 and 13-15 are the antibiotic cocktail (Abx) treatment durations; week 9 is FMT1 and week 15 is FMT2. Due to the presence of antibiotic cocktails in the drinking water, which have a bitter taste, it takes the mice time to adjust to the taste. Furthermore, the reduction of the intestinal microbiome leads to a decreased ability to absorb food, resulting in significant weight loss throughout the treatment period, which explains the sharp drop in weight gain after weeks 7 and 13. Fig. 5B. Mice diastolic and systolic echo parameters at the midpoint of the experiment, approximately one month after the first Fecal Microbiota Transplant (FMT 1). Fig. 5C. Mice diastolic and systolic echo parameters at the endpoint of the experiment, approximately one month after the second Fecal Microbiota Transplant (FMT 2). For Figs. 5B, 5C: Upper left: E / e’ ratio; upper right: e' Wave; lower left: LV-GLS; and lower right: LV-EF. Fig. 5D. Quantification of positive cells per tissue in myocardium sections from mice strained with immunofluorescent antibodies against CD4, CD8, CD68, and CD31, identifying helper T cells, cytotoxic T cells, macrophages, and endothelial cells, respectively. Upper left panel: CD8; upper right panel: CD4; lower panel left: CD31, lower panel right: CD68. Fig. 5E shows sPLS-DA analysis of metabolic profile derived from humanized microbiota mice plasma before (upper left panel), after (upper right panel), and before vs. after (lower panel) the second antibiotics cocktail treatment (Abx). Fig. 5F shows five downregulated metabolites that were unique to the obese-HFpEF FMT mice group. Fig. 5G shows one upregulated metabolite was unique to the obese-HFpEF FMT mice group (upper panel).

[0069] DETAILED DESCRIPTION OF THE INVENTION

[0070] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0071] The present invention relates to treatments of Heart Failure with Preserved Ejection Fraction (HFpEF) by fecal microbiota transplant (FMT), and / or by metabolites (a single metabolite or a combination of metabolites) which are altered in this condition. The treatments are based on analyses of FMT from human subjects using a “two-hit” mouse model of HFpEF, wherein the FMT is from healthy or obese human subjects not diagnosed with HFpEF, or from human subjects diagnosed with HFpEF.

[0072] Generating and characterizing the two-hit HFpEF mouse model

[0073] The two-hit mouse model of HFpEF was generated by feeding mice with a combination of N“-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO biosynthesis inducing hypertension, and a high fat diet (HFD), causing obesity. Echocardiographic assessment of diastolic parameters including e' Wave, E / e’ ratio, left ventricle (LV) global longitudinal strain (GLS), and LV Ejection fraction (EF) from m-mode, showed impaired diastolic function and increased filling pressures (Fig. IB), in the HFpEF mouse model.

[0074] As shown in Fig. ID (lower panel), the gut microbiota composition was significantly different between the obese group fed with HFD (HFD and HFpEF groups) and non-obese group fed with standard CHOW diet (CHOW and L-NAME groups), but more importantly, the obese HFpEF group had a significantly distinct microbial composition compared with the obese non- HFpEF group both in humans (lower panel, Obese-Control) and in mice (lower panel, HFD), highlighting bacterial populations specific to HFpEF. Exploring the relative abundance, it was found that the relative abundance of two families of Bacteroidales (Bacteroidales bacleroidctceae. Bacteroidales Prevotellaceae) was increased in the HFpEF groups as compared to the obese non- HFpEF group in humans and consistently in the mouse model. It was further found that the families Clostridiales Ruminococcaceae and Clostridiales Lachnospiraceae were decreased in the HFpEF group in both human and mouse, (not shown).

[0075] Simulating HFpEF in the mouse model by FMT

[0076] Experiments involving Fecal Microbiota Transplant (FMT) from obese-HFpEF patients, obese-only patients (non-HFpEF), and controls into mice of the two-hit model, in which their gut microbiome had been abolished by administering an antibiotic cocktail prior to the FMT, were conducted, to generate obese-HFpEF, obese-only, and control FMT mouse models, respectively. It was found that the obese-HFpEF humanized gnotobiotic mice developed significantly more severe myocardial disease, including worsening diastolic dysfunction by echocardiography (Fig. 2B), and increased endothelial dysfunction by histological analysis of the myocardium (Fig. 2C), as compared to the obese-only and control humanized gnotobiotic mice.

[0077] Completed metabolomic analyses of cardiac tissues and plasma samples from these mice after FMT from human samples were performed, which revealed changes in pathways, genes and metabolites which were specific to gut microbiota of obese HFpEF phenotype as compared to obese-only phenotype (Figs. 3A-3F). These molecules may serve as pivotal links connecting gut dysbiosis to the pathogenesis of obesity related HFpEF, holding significant promise for future therapeutic and diagnostic advancements. Treating HFpEF model mice with FMT from healthy individuals

[0078] The possibility of improving cardiac diastolic function of the humanized microbiota mice following FMT with human microbiome from obese HFpEF patients, by subsequently receiving additional FMT from healthy individuals, was also examined. Diastolic function by echocardiography was significantly improved in mice subsequently treated with FMT from healthy individuals, compared to those mice who received additional FMT from HFpEF patients. In contrast, those mice who received the first FMT from healthy subjects followed by FMT from HFpEF patients showed worsening of the diastolic function (Figs. 5B-5H). These findings indicate that diastolic function is dynamic and can be improved with FMT from healthy individuals, suggesting that gut microbiota manipulation can potentially be a therapeutic target for HFpEF.

[0079] Methods of treating HFpEF

[0080] In some embodiments, the present invention provides a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, by administering to the subject a composition including gut bacterial populations. Alternatively or in addition, the method of treating HFpEF in a subject in need thereof may be through the administration of metabolites which are missing in the HFpEF subject compared to a subject not having HFpEF, or agents which reduce the levels of gut bacterial populations and / or metabolites which are present in the HFpEF subject in excess compared to a subject not having HFpEF.

[0081] In some embodiments, there is provided a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, including administering to the subject at least one composition including: a) fecal material from a subject not diagnosed with HFpEF; b) a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c) at least one antibiotic agent capable of reducing the abundance or activity of gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d) at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and / or e) at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0082] In some embodiment, the at least one composition is a single composition. In some embodiments, the at least one composition is more than one composition, such as 2, 3, 4, or 5 compositions, and each of the more than one compositions includes any combination of one or more of (a), (b), (c), (d), and (e) or any combination of components thereof. In some embodiments, each of the more than one compositions includes different elements selected from (a), (b), (c), (d), and (e), such that any element is present in no more than one composition.

[0083] In some embodiments, the gut-associated bacteria are anaerobic bacteria.

[0084] Administering fecal material

[0085] In some embodiments, the at least one composition includes fecal material from a subject not diagnosed with HFpEF.

[0086] The term “subject not diagnosed with HFpEF”, as used here, also means a subject who is not suffering from HFpEF. The subject need not necessarily be completely healthy. In some embodiments, the subject is obese. In some embodiments, the subject is not obese.

[0087] The subject in need of treatment is a subject diagnosed with HFpEF. In some embodiments, the subject in need of treatment is obese. In some embodiments, the subject in need of treatment has at least one symptom associated with metabolic syndrome, such as increased weight, high blood pressure, diabetes, etc. In some embodiments, the subject in need of treatment is suffering from a disease or disorder selected form obesity, insulin resistance, type 2 diabetes, hypertension, and dysbiosis.

[0088] The term “fecal material” relates to material obtained from human feces or stools, or any portion thereof which includes gut bacteria, or gut microbiota.

[0089] In some embodiments, the fecal material is a fresh fecal material. In some embodiments, the fecal material has been cryopreserved.

[0090] The term “fresh” with respect to fecal material, means fecal material that has not been frozen or cryopreserved. In some embodiments, time passed since defecation of the fecal material is not more than 6, 8, 10, 18, or 24 hours.

[0091] In some embodiments, the composition further includes saline. In some embodiments, the composition further includes a cryoprotectant including glycerol and / or polyethylene glycol (PEG). In some embodiments, the concentration of the PEG in the composition is about 10-100, 20-100, 30-100, 30-90, 30-80, or about 40-60 gr / L. In some embodiments, the concentration of the glycerol in the composition is about 5%-20% or 5%-l 5%, or about 10%.

[0092] In some embodiments, the fecal material is administered at a therapeutically effective amount.

[0093] In some embodiments, the fecal material is administered together with isolated gut- associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0094] In some embodiments, the fecal material is administered together with at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus.

[0095] In some embodiments, the fecal material is administered together with at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0096] In some embodiments, the fecal material is administered together with at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0097] Administering gut-associated bacteria

[0098] As demonstrated and explained below (see Figs. 3B-3C), certain gut microbiome populations have been found to be under-represented in HFpEF patients compared to non-HFpEF subjects such as obese patients, and therefore administering these gut microbiome populations may help treat the HFpEF. Especially relevant for such treatment are gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance. Nonlimiting examples include Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

[0099] In some embodiments, the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae,

[0100] Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

[0101] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from at least one family selected from Bacteroidaceae,

[0102] Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae,

[0103] Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0104] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Bacteroidaceae.

[0105] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Erysipelotrichaceae.

[0106] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Lachnospiraceae.

[0107] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Odoribacteraceae.

[0108] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Porphyromonadaceae.

[0109] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Ruminococcaceae.

[0110] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Muribaculaceae (S24-7).

[0111] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from Rikenellaceae.

[0112] In some embodiments, the at least one composition includes a therapeutically effective amount of isolated gut-associated bacteria from at least one genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0113] In some embodiments, the composition includes a therapeutically effective amount of at least one gut-associated bacterial species selected from Parabacteroides distasonis and Clostridium aldenense.

[0114] In some embodiments, the composition includes a therapeutically effective amount of at least 2, 3, 4, 5, 6, or 7 bacterial families selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0115] In some embodiments, the composition includes a therapeutically effective amount of at least 2, 3, 4, 5, 6, or 7 bacterial genera selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0116] In some embodiments, the fecal material includes a therapeutically effective amount of gut- associated bacteria belonging to at least one bacterial family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0117] In some embodiments, the fecal material includes a therapeutically effective amount of gut- associated bacteria belonging to at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

[0118] The term “gut-associated bacteria” relates to bacterial populations which are found in human gut microbiome. The bacterial populations are not necessarily directly obtained from fecal material and may be obtained from other sources, for example, from laboratory cultures. The term “isolated gut-associated bacteria” relates to gut-associated bacteria which are not part of fecal material.

[0119] In some embodiments, the isolated gut-associated bacteria are isolated from fresh fecal samples. In some embodiments, the isolated gut-associated bacteria are isolated from a cryopreserved fecal sample. In some embodiments, the isolated gut-associated bacteria are cultured in a laboratory in vitro.

[0120] In some embodiments, the composition includes a combination of fecal material and isolated gut-associated bacteria.

[0121] The term “therapeutically effective amount” relates to the amount of the bacteria or of fecal material needed to have a therapeutic effect. This amount is generally related to the type of bacteria, type of symptoms, and the severity of the disease.

[0122] In some embodiments, the composition is administered by rectal administration. In some embodiments, the composition is administered by oral administration.

[0123] In some embodiments, the isolated gut-associated bacteria are administered by rectal administration, such as by enema or suppositories. In some embodiments, the isolated gut- associated bacteria are administered orally.

[0124] In some embodiments, the method further includes a step of administering antibiotics prior to administering the composition.

[0125] In some embodiments, the method further includes a step of administering antibiotics after administering the composition.

[0126] In some embodiments, the antibiotics is selected from vancomycin, neomycin, metronidazole, ampicillin, fidaxomicin, rifaximimin, linezolid, daptomycin, quinupristin- dalfopristin, tigecycline, ceftaroline, ceftobiprole, televancin, teicoplanin, dalbavancin, metronidazole, ortivancin, telavancin, oritavancin, iclaprim, plazomicin, amikacin, tinidazole, secnidazole, amoxicillin, piperacillin, ceftriaxone, surotomycin, bezlotoxumab, rifamycin SV MMX, nitazoxanide, tedizolid, eravacycline, omadacycline, cefiderocol, and a combination thereof.

[0127] In some embodiments, the gut-associated bacteria is administered together with fecal material.

[0128] In some embodiments, the gut-associated bacteria is administered together with at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus.

[0129] In some embodiments, the gut-associated bacteria is administered together with at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0130] In some embodiments, the gut-associated bacteria is administered together with at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0131] Administering antibiotic agents

[0132] As demonstrated and explained below (see Fig. 3B-3C), certain gut microbiome populations have been found to be over-represented in HFpEF patients compared to non-HFpEF subjects, and therefore reducing the levels of these populations may help treat the HFpEF. Especially relevant populations include gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction. Nonlimiting examples include the families Alcaligenaceae (e.g., Sutterella) and Lachnospiraceae.

[0133] In some embodiments, the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

[0134] In some embodiments, the at least one composition includes at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus.

[0135] In some embodiments, the at least one composition includes at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus

[0136] In some embodiments, the at least one composition includes at least one antibiotic agent capable of reducing a level of Sutterella genus.

[0137] In some embodiments, the at least one composition includes at least one antibiotic agent capable of reducing a level of Lachnospiraceae genus.

[0138] In some embodiments, the Lachnospiraceae genus is not Blautia or Clostridium. In some embodiments, the Lachnospiraceae genus is not Blautia. In some embodiments, the Lachnospiraceae genus is not Clostridium.

[0139] In some embodiments, the at least one antibiotic agent is administered together with fecal material.

[0140] In some embodiments, the at least one antibiotic agent is administered together with isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0141] In some embodiments, the at least one antibiotic agent is administered together with at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0142] In some embodiments, the at least one antibiotic agent is administered together with at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0143] Administering metabolites or blocking agents

[0144] Based on Figs. 3A-3F (Example 2), certain metabolites, specifically amino acid and shortchain carboxylic acid metabolites, which are associated with the gut microbiome changes are decreased in the HFpEF subjects. Therefore, administering these metabolites to patients is expected to ameliorate the symptoms. The metabolite may be a host metabolite or a bacterial metabolite. Nonlimiting examples include 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy- D-proline, and phenylacetylglycine.

[0145] In some embodiments, the metabolite is selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

[0146] In some embodiments, the at least one composition includes at least one metabolite selected from tryptophan derived metabolites; monounsaturated fatty acids derived metabolites; amino acids, peptides, and analogues derived metabolites; D-Amino acid metabolism derived metabolites; and phenylalanine metabolism derived metabolites.

[0147] Tryptophan derived metabolites may include tryptamine, kynurenine and indolic compounds, including indole-3 -aldehyde (lAld), indole-3 -acetic-acid (IAA) and indole-3 - propionic acid (IP A), and other indolic compounds.

[0148] Monounsaturated fatty acids derived metabolites may include 2-Butenoic acid.

[0149] Amino acids, peptides, and analogues derived metabolites may include N-acetylmethionine, cis-4-Hydroxy-D-proline, and Phenylacetylglycine (PAG).

[0150] D-Amino acid metabolism derived metabolites may include cis-4-Hydroxy-D-proline.

[0151] Phenylalanine metabolism derived metabolites may include Phenylacetylglycine (PAG).

[0152] In some embodiments, the at least one composition includes at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine. In some embodiments, the at least one composition includes 2-Butenoic acid.

[0153] In some embodiments, the at least one composition includes N-acetylmethionine.

[0154] In some embodiments, the at least one composition includes cis-4-Hydroxy-D-proline.

[0155] In some embodiments, the at least one composition includes phenylacetylglycine.

[0156] In some embodiments, the at least one composition includes at least 2, 3, 4, or 4 metabolites selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0157] In some embodiments, the at least one metabolite is administered together with fecal material.

[0158] In some embodiments, the at least one metabolite is administered together with isolated gut- associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0159] In some embodiments, the at least one metabolite is administered together with at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus.

[0160] In some embodiments, the at least one metabolite is administered together with at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0161] Based on Figs. 3A-3F (Example 2), certain small molecule metabolites which are associated with the gut microbiome changes, and particularly with redox regulation, nucleotide metabolism, and microbial energy-associated pathways, are increased in the HFpEF subjects. The metabolite may be a host metabolite or a bacterial metabolite. Nonlimiting examples for such small molecule metabolites include deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid. Therefore, administering agents which block production or activity of these small molecule metabolites to patients is expected to ameliorate the symptoms.

[0162] In some embodiments, the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0163] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of a metabolite selected from purine metabolism derived metabolites; cAMP signaling and mTOR signaling pathways derived metabolites; ascorbate and aldarate metabolism derived metabolites; glyoxylate and dicarboxylate metabolism derived metabolites; and microbial metabolism in diverse environments derived metabolites.

[0164] Purine metabolism derived metabolites may include deoxyguanosine diphosphate (dGDP), adenosine monophosphate (AMP), and adenylsuccinic acid. cAMP signaling and mTOR signaling pathways derived metabolites may include adenosine monophosphate (AMP).

[0165] Ascorbate and aldarate metabolism derived metabolites may include L-Threonic acid.

[0166] Glyoxylate and dicarboxylate metabolism derived metabolites may include mesaconic acid.

[0167] Microbial metabolism in diverse environments derived metabolites may include mesaconic acid.

[0168] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L- Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0169] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of deoxyguanosine diphosphate (dGDP).

[0170] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of L-Threonic acid.

[0171] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of adenyl succinic acid.

[0172] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of adenosine monophosphate (AMP).

[0173] In some embodiments, the at least one composition includes at least one agent that blocks production or activity of mesaconic acid.

[0174] In some embodiments, the composition includes at least 2, 3, 4, or 5 agents that block production or activity of at least 2, 3, 4, or 5 metabolites selected from deoxy guanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0175] In some embodiments, at least one agent that blocks production or activity of a metabolite is administered together with fecal material.

[0176] In some embodiments, at least one agent that blocks production or activity of a metabolite is administered together with isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0177] In some embodiments, at least one agent that blocks production or activity of a metabolite is administered together with at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus.

[0178] In some embodiments, at least one agent that blocks production or activity of a metabolite is administered together with at least one metabolite selected from 2-Butenoic acid, N- acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0179] In some embodiments, the method includes administering a combination of two or more of the compositions described herein. In some embodiments, the method includes administering a composition including fecal material in combination with at least one composition including isolated gut-associated bacteria.

[0180] The administration may be by the same route or by different routes. For examples, one of the compositions may be administered orally and another may be administered rectally.

[0181] In some embodiments, the two or more compositions may be administered together (i.e., at the same time, simultaneously). In some embodiments, the two or more compositions may be administered at different times.

[0182] Compositions and compositions for use in treating HFpEF

[0183] In some embodiments, the present invention provides a composition including at least one of: a. fecal material from a subject not diagnosed with HFpEF; b. a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c. at least one antibiotic agent capable of reducing the abundance or activity of gut- associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d. at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and e. at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0184] In some embodiments, the present invention provides a composition for use in a method of treating heart failure with preserved ejection fraction (HFpEF), the composition including at least one of: a. fecal material from a subject not diagnosed with HFpEF; b. a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c. at least one antibiotic agent capable of reducing the abundance or activity of gut- associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d. at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and e. at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0185] In some embodiments, the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are anaerobic bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

[0186] In some embodiments, the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

[0187] In some embodiments, the metabolite is selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

[0188] In some embodiments, the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0189] In some embodiments, the Lachnospiraceae genus is not Blautia or Clostridium. In some embodiments, the Lachnospiraceae genus is not Blautia. In some embodiments, the Lachnospiraceae genus is not Clostridium.

[0190] In some embodiments, the composition or the composition for use includes any combination of (a), (b), (c), (d), and (e), or any combination of components included therein.

[0191] In some embodiments, the composition or the composition for use includes fecal material from a subject not diagnosed with HFpEF.

[0192] In some embodiments, the composition or the composition for use includes isolated gut- associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0193] In some embodiments, the composition or the composition for use includes at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus. In some embodiments, the composition or the composition for use includes at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0194] In some embodiments, the composition or the composition for use includes at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0195] In some embodiments, the composition or the composition for use includes fecal material from a subject not diagnosed with HFpEF and isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0196] In some embodiments, the composition or the composition for use includes fecal material from a subject not diagnosed with HFpEF and at least one antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus.

[0197] In some embodiments, the composition or the composition for use includes fecal material from a subject not diagnosed with HFpEF and at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine.

[0198] In some embodiments, the composition or the composition for use includes fecal material from a subject not diagnosed with HFpEF and at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0199] The above embodiments for the composition also apply to the combination and the combination for use below.

[0200] In some embodiments, the present invention provides a combination including at least two compositions selected from: a. a composition including fecal material from a subject not diagnosed with HFpEF; b. a composition including a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c. a composition including at least one antibiotic agent capable of reducing the abundance or activity of gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d. a composition including at least one metabolite selected from an amino acid, a shortchain carboxylic acid, and derivative thereof; and e. a composition including at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0201] In some embodiments, the present invention provides a combination for use in a method of treating heart failure with preserved ejection fraction (HFpEF), the combination including at least two compositions selected from: a. a composition including fecal material from a subject not diagnosed with HFpEF; b. a composition including a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c. a composition including at least one antibiotic agent capable of reducing the abundance or activity of gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d. a composition including at least one metabolite selected from an amino acid, a shortchain carboxylic acid, and derivative thereof; and e. a composition including at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

[0202] In some embodiments, the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are anaerobic bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

[0203] In some embodiments, the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

[0204] In some embodiments, the metabolite is selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

[0205] In some embodiments, the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0206] Definitions and embodiments mentioned above and which may be relevant to the composition or the use embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

[0207] In some embodiments, any of the compositions disclosed herein, or combinations thereof, further include a pharmaceutically acceptable carrier.

[0208] The composition of the present invention may be formulated in any conventional manner using one or more physiologically or pharmaceutically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition, not being deleterious to the recipient thereof, and not significantly interfering with the activity of the polypeptide of the invention, or of any other active ingredient in the composition. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the composition may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0209] The term “treating” or “treatment”, as used herein, refers to means of obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease or condition and / or symptoms attributed to the disease or condition. The term includes inhibiting the disease or condition, i.e. arresting its development; or ameliorating the disease or condition, i.e. causing regression of the disease or condition, e.g., by eliminating or ameliorating its symptoms.

[0210] Suitable modes of administration of the compositions disclosed herein include, but are not limited to parenteral administration such as intravenous, subcutaneous, intramuscular, and intrathecal; oral administration; rectal administration such as by enema or suppositories; or topical administration.

[0211] The compositions mentioned herein may be prepared for administration in appropriate dosage forms, including enema, tablet, pill, capsule, solution for injection, etc.

[0212] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0213] The term "a" and "an" refers to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0214] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0215] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the scope of the present invention as described by the claims, which follow.

[0216] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0217] EXAMPLES

[0218] Materials and Methods

[0219] Experimental design

[0220] Animals

[0221] This study used male C57BL / 6J mice aged 8 to 12 weeks (Harlan; Ein Kerem, Israel). Mice were kept in a pathogen-free unit under controlled 12-h light / 12-h dark cycle and an ambient temperature of 21 ± 1°C and humidity of 40%-50%. All experimental procedures were approved by the Hebrew University Animal Care and Use Committee (MD-20-16160-5 and MD-20-16703- 5) and performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85- 23).

[0222] The two-hit mouse model

[0223] HFpEF was elicited in mice by concomitant metabolic stress (obesity and metabolic syndrome) and mechanical stress (hypertension) using a combination of high-fat diet (HFD) (60% kilocalories from fat, Envigo) and Nw-nitro-L-arginine methyl ester (L-NAME), a consecutive NO synthase inhibitor, (0.5 g I-1in drinking water after adjusting the pH to 7.40, Sigma-Aldrich), both administered for 10 weeks. This “two-hit” mouse model of HFpEF was previously shown to recapitulate various systemic and cardiovascular features of HFpEF in humans (Schiattarella et al. Nitrosative stress drives heart failure with preserved ejection fraction. Nature. 2019;568:351-356).

[0224] The humanized HFpEF microbiota mouse model

[0225] Human fecal material was treated as follows: 1 g of feces was added to a 50-mL sterile plastic container, and 5 mL of physiological saline (0.9% w / v of NaCl) were added to the container. The fecal material was suspended using a spatula, and 1.25 mL of sterile 50% glycerol was added (to a final concentration of 10% glycerol). The mixture was suspended with a spatula and frozen at -80°C for storage.

[0226] In order to obtain a humanized HFpEF microbiota mouse model, after 7 weeks of HFD and L-NAME treatment, mice received an antibiotic cocktail (Abx) ad libitum in their drinking water for two weeks before the fecal microbiome transplantation (FMT).

[0227] For the FMT procedure, 200 mg of frozen feces per recipient were suspended in sterile PBS (-Ca2+, -Mg2+) under sterilized conditions, vortexed until dissolved, and allowed to precipitate for 10 minutes. Recipient mice were administered 200 pl of the supernatant solution by oral gavage. After FMT, all mice were housed in a clean pathogenic room in sterilized or autoclaved cages to minimize unwanted exogenous colonization with exogenous bacteria.

[0228] Antibiotic cocktail (Abx)

[0229] The antibiotic cocktail administered to the mice was composed of vancomycin hydrochloride (0.5g / l), neomycin trisulfate salt hydrate ( 1 g / 1), metronidazole ( 1 g / 1), and ampicillin sodium ( 1 g / 1).

[0230] Conventional echocardiography and Doppler imaging

[0231] Transthoracic echocardiography was performed using a VisualSonics Vevo 3100 system equipped with an MX550D transducer (Visual Sonics). The left ventricular ejection fraction was obtained from long-axis B-mode scans to assess left ventricular systolic function. Other indices of systolic function were obtained from short-axis M-mode scans at the midventricular level, as indicated by the presence of papillary muscles, in anesthetized, gently restrained mice. Apical four- chamber views were obtained in anesthetized mice for diastolic function measurements using pulsed-wave and tissue Doppler imaging at the level of the mitral valve. Anesthesia was induced by 2-3% isoflurane and confirmed by a lack of response to firm pressure on one of the hind paws. During echocardiogram acquisition, performed under body-temperature-controlled conditions, isoflurane was reduced to 0.5-1.5% and adjusted to maintain a heart rate in the range of 400-500 beats per minute. Parameters collected included: heart rate, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular mass, left ventricular ejection fraction, peak Doppler blood inflow velocity across the mitral valve during early diastole, peak tissue Doppler myocardial relaxation velocity at the mitral valve annulus during early diastole, and early filling deceleration time. At the end of the procedures, all mice recovered from anesthesia without difficulties. All parameters were measured at least nine times (3 times for every three different videos) and presented as means ± standard error of the mean (SEM).

[0232] Speckle-tracking echocardiography and strain analysis

[0233] B-mode traces acquired from the parasternal long-axis view were used to calculate global strain in longitudinal dimensions using Vevo Strain software (Visual Sonics) and a speckletracking algorithm. The values generated by strain analysis in the longitudinal dimension are indicative of fiber shortening. B-mode images were selected based on quality (high frame rates) and on the ability to visualize both the endocardial and epicardial left ventricular wall borders. Borders of the endocardium and epicardium were traced, and semi -automated strain analysis was performed. Average peak global strain values were obtained from at least six independent anatomical segments of the left ventricle.

[0234] Histology

[0235] Mice cardiac ventricles were collected and fixed in 4% paraformaldehyde overnight and processed for routine paraffin histology (5-pm sections), then stained with Masson’s trichrome. For immunofluorescence, paraffin sections were deparaffinized and exposed to antigen retrieval using hot citrate buffer (Antigen Unmasking Solution, Vector Laboratories). The sections were then blocked with 3% bovine serum albumin (BSA) in PBS with 0.1% Tween™ 20 (PBS-T) and incubated with the following primary antibodies: rabbit polyclonal anti-CD31 antibody (1 :1000; Abeam, abl24432), rabbit monoclonal anti-CDl lb antibody (1 :500; Abeam, abl33357), rabbit anti-CD68 monoclonal antibody (l;100; Abeam, ab283654), and rat anti-CD3 monoclonal antibody (l;100; Abeam, abl 1089) overnight at 4°C. After washing, sections were incubated with secondary Cy5-conjugated donkey anti-rabbit IgG (1 : 100 in CAS-Block, Jackson Immunoresearch Laboratories) and Cy5-conjugated donkey anti-rat IgG (1 :100 in CAS-Block, Jackson Immunoresearch Laboratories) for 1 hour at room temperature. Sections were then mounted using anti -fade reagent supplemented with DAPI (EMS) (Cat# 17984-24, Biotrend), and images were obtained using Nikon fluorescent microscopy.

[0236] Cytokine biomarkers analysis

[0237] Concentrations of tumor necrosis factor-alpha (TNF-a), Interleukin- ip (IL-ip), IL-6, Interferon-y (IFN-y), IL- 17 were simultaneously quantified in peripheral plasma samples with the Milliplex Mouse Cytokine / Chemokine Magnetic Bead Panel (Merck -Millipore) using Luminex technology according to the manufacturer’s instructions. 16S rDNA sequencing

[0238] Fresh fecal pellets were aseptically collected into sterile tubes and stored at -80°C. DNA was isolated from fecal samples using a PureLink Microbiome DNA Purification Kit (Invitrogen). PCR amplification was performed on the V3 and V4 regions of the 16S rDNA gene using the 34 IF and 805R primers (Herlemann et al., 2011, ISME Journal, 5, 1571-1579). Sequencing of these amplicons was performed on an Illumina MiSeq sequencer using 2 x 250-bp paired-end reads.

[0239] 16S rDNA analysis

[0240] Microbiome bioinformatics analyses were performed with QIIME 2, a plugin-based system that, in some cases, wraps other microbiome analysis methods, as previously described (Bolyen et al., Nat Biotechnol. 2019;37:852-857). Briefly, de-multiplexed FASTQ files were obtained from the sequencing facility. PE FASTQ files were further processed for adapter sequence removal using the Cutadapt trim-paired plugin, followed by denoising with DADA2 (via q2-dada2) to identify all observed amplicon sequence variants (ASVs). All ASVs were aligned with mafft (via q2- alignment) and used to construct a phylogeny with fasttree2 (via q2 -phylogeny). Taxonomy was assigned to ASVs using the q2 -feature-classifier classify-skleam naive Bayes taxonomy classifier, which was pre-trained against sequences extracted from Greengenes 13 8 99% operational taxonomic unit (OUT) sequences using primers set 341F-805R. Qiime2 object table. qza was exported into biom format and feature_table.txt, Qiime2 object phylogenic rooted- tree. qza was exported to Newick tree format Qiime2 object taxonomy. qza was exported to taxonomic_feature_table.txt. The following files were used as inputs to the Microbiome Analyst: comprehensive statistical, visual and meta-analysis of microbiome online tool, after minor manual format editing, along with a metadata file which describes the biological history of each one of the samples in the dataset (metadata file is an edited table based on the QIIME2 manifest file), as previously described. (Chong et al., Nat Protoc. 2020;15:799-821).

[0241] Total RNA sequencing

[0242] 100 mg of ventricle tissue from all mouse groups (with and without FMT) was dissected and homogenized in 1 ml of TRI reagent (Sigma) at room temperature. Subsequently, 100 ul of 1- bromo-3 -chloropropane (Sigma) was added, and samples were incubated for 15 min at room temperature and centrifuged at 12000 x g for 15 min at 2°C. Subsequently, 500 ul of 2-propanol (Sigma) was added, and samples were incubated for 10 min at room temperature and centrifuged at 12000 x g for 10 min at 2 °C. The supernatant was removed, and the RNA pellet was washed by adding 1 ml of 75% ethanol in DEPC-treated water (Sigma), and samples were centrifuged at 12000 x g for 5 min at 2 °C. The ethanol was dried, and the samples were incubated for 10 minutes at room temperature. Subsequently, 25 ul of DEPC water (Sigma) was added, and samples were incubated for 15 min at 55-60°C and mixed by pipetting.

[0243] Total RNA sequencing data analysis

[0244] Trimming and filtering of raw reads:

[0245] The NextSeq basecalls files were converted to fastq files using the bcl2fastq program (v2.20.0.422) with default parameters (without trimming or filtering applied at this stage). Raw reads (fastq files) were inspected for quality issues with FastQC (v0.11.8). Following that, the reads were quality -trimmed with cutadapt (v2.10), using a quality threshold of 32 for both ends; poly-G sequences (NextSeq’s no signal) and adapter sequences were removed from the 3' end; and poly-T stretches were removed from the 5' end (being the reverse complement of poly-A tails). The Cutadapt parameters included using a minimal overlap of 1, allowing for read wildcards, and filtering out reads that became shorter than 15 nt. Finally, low-quality reads were filtered out using fastq quality filter (v0.0.14, FASTX package), with a quality threshold of 20 at 90 percent or more of the read's positions.

[0246] Alignment and counting:

[0247] The processed reads were aligned with TopHat (v2.1.), allowing for 5 mismatches (see full command below). The reference genome included the mouse GRCm39, with annotations from Ensembl release 106. Quantification was done with htseq-count (vO.12.4). Strand information was set to'reverse', and an annotation file that included the human genes but lacked information for genes of type IG, TR, artifact, miRNA, Mt_rRNA, Mt_tRNA, ncRNA, piRNA, pre_miRNA, rRNA, ribozyme, sRNA, scRNA, scaRNA, siRNA, snRNA, snoRNA, tRNA, and vaultRNA was used. Counts of technical replicates were merged into one sample.

[0248] Differential expression:

[0249] Normalization and differential expression analysis were performed using the DESeq2 package (vl.22.2). Genes with a sum of counts less than 10 over all samples were filtered out, and then size factors and dispersion were calculated. Normalized counts were used for several quality control assays, such as count distributions and principal component analysis, which were calculated and visualized in R (v3.6.3). Pair-wise comparisons were tested with default parameters (the Wald test), except not using the independent filtering algorithm. The significance threshold was chosen as padj<0.1 (default). In addition, significant genes were further filtered by the log2FoldChange value. This filtering was baseMean-dependent and required a baseMean above 5 and an absolute log2FoldChange higher than 5 / sqrt(baseMean) + 0.1 (for highly expressed genes, this means a requirement for a fold change of at least 1.2, while genes with a very low expression would need a 4.5-fold change to pass the filtering). While the genes that passed the default (padj<0.01) filter are termed "Sig,” the subset of genes that also passed the additional filter are termed "Best." Finally, results were combined with gene details (such as symbols, known transcripts, etc.), taken from the results of a BioMart query (Ensembl, release 106), to produce the final Excel file.

[0250] Metabolomics

[0251] Heart tissue and blood samples were collected immediately after sacrificing into Eppendorf and heparin tubes, respectively. Plasma was collected after 4000 rpm for 10 min at 4°C. The heart tissue was frozen in liquid nitrogen and stored at -80°C until analysis. Metabolites were determined based on liquid chromatography mass spectrometry (LC-MS) technology using a high-resolution accurate mass instrument (Q-Exactive Plus, Thermo Scientific) coupled with ultra-performance liquid chromatography (UPLC) systems (Thermo Scientific), for untargeted metabolism profiling analysis. The metabolomic analysis and the pathway analysis were done by the web interface of “MetaboAnalyst” (JavaServer Faces Technology using the PrimeFaces library (vl3.0)).

[0252] Statistics

[0253] Continuous data are presented as mean ± SEM and checked for normality using D'Agostino- Pearson Omnibus K-squared or Shapiro-Wilk test for small sample size. Differences were analyzed by 2-tailed Student's t-test or one-way analysis of variance (ANOVA) followed by post hoc Tukey’s honestly significant difference tests for multiple pairwise comparisons, unless otherwise stated. For comparisons, a P value of <0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism software version 10.0 (Graph-Pad Software). Experimental mice were randomly assigned to each experimental or control group. Investigators were blinded to the treatments of the individual animals during the experiments and outcome assessment.

[0254] Example 1: Characterization of the two-hit mouse model

[0255] To establish and characterize the two-hit mouse model, four groups of mice were studied (n = 6-8 / group), and all mice were sacrificed 10 weeks after the study initiation. The groups included:

[0256] 1) CHOW / control group: mice fed with standard chow (Teklad);

[0257] 2) N(co)-nitro-L-arginine methyl ester (L-NAME) group: mice fed with standard chow and treated with L-NAME (0.5 g 1-1, Sigma-Aldrich) in the drinking water; 3) high-fat diet (HFD) group: mice fed with HFD (60% kilocalories from fat, Envigo); and

[0258] 4) HFpEF group (two-hit): mice fed with HFD and treated with L-NAME for 10 weeks to develop HFpEF.

[0259] Echo measurements were made at the baseline and at the end of the experiment (Fig. IB).

[0260] Inflammatory fibrosis of the myocardium was evaluated by staining with antibodies against inflammatory markers: CD 11b, a CD8 cytotoxic T cell activation marker, and CD31, a vascular differentiation marker for endothelial cells, and by visually evaluating scarring by staining for fibrotic areas (Fig. 1C).

[0261] A Principal Coordinates Analysis (PCoA) of the gut microbiome is shown in Fig. ID, comparing samples from humans and mice groups and showing the diversity of the populations.

[0262] Finally, a comparison of the relative abundance of bacterial populations between the different groups in humans and in mice was conducted. The comparison showed that some populations, e.g., Bacteroidales bacleroidctceae. Bacteroidales Prevotellaceae, were found to be expanded in the HFpEF groups, while other populations, e.g., Clostridiales Ruminococcaceae and Clostridiales Lachnospiraceae , were decreased in the HFpEF groups.

[0263] Example 2: Humanized HFpEF microbiota mouse model

[0264] Donors for Fecal Microbiota Transplant (FMT) included 20 obese-HFpEF human subjects, 10 obese (non-HFpEF) human subjects and 10 control (non-obese, non-HFpEF) human subjects.

[0265] Five groups of mice (n = 10 / group) were treated with L-NAME only until week 7 and fed with HFD throughout the experiment. Mice were administered an antibiotic cocktail (Abx) for 2 weeks (weeks 7-9), and FMT from obese-HFpEF donors, obese-only donors, or control donors was performed at week 9 to generate the humanized HFpEF microbiota mouse model. Echo measurements were taken before the FMT and one month after the FMT (Fig. 2B).

[0266] Myocardium sections from the three groups of the FMT mice were stained with antibodies against CD3, CD68, and CD31 (identifying T cells, macrophages, and endothelial cells, respectively) (Fig. 2C), showing increased lymphocytes and macrophages in myocardial tissue form the obese-HFpEF FMT mice. Further evidence for the increased inflammatory activity in the obese-HFpEF FMT mice is shown by increased levels of the inflammatory cytokines IFNy, IL- 10, IL- 17, and TNFa in their plasma (Fig. 2D).

[0267] A comparison of gene expression from FMT obese-HFpEF mice and FMT obese mice shows upregulation or downregulation of certain genes from certain metabolic pathways in the HFpEF mice (Fig. 3A). In addition, microbiome from all test groups was sequenced and analyzed to show the diversity and main families representing the difference between the populations, in other words the beta-diversity of the microbial communities which illustrates the relative differences in the different treatments (Figs. 3B-3C). The main populations under -represented in the HFpEF mice compared to the obese-only mice were from the genera Bacteroidaceae Bacteroides, Erysipelotrichaceae Allobaculum, Lachnospiraceae Blautia, Lachnospiraceae Clostridium (and species aldenense), Odoribacteraceae Odoribacter, Porphyromonadaceae Parabacteroides (species distasonis), and Ruminococcaceae Oscillospira, and the families Muribaculaceae (S24-7) and Rikenellaceae, while the main populations over -represented in the HFpEF mice compared to the obese-only were Lachnospiraceae and Alcaligenaceae Sutterella.

[0268] Next, metabolomics profiles analyses and pathway analyses were conducted on the humanized microbiota model mice plasma and heart tissue (Figs. 3D-3F), comparing the obese group to the obese-HFpEF group. Metabolites that were found to be lower in the HFpEF group included: tryptophan derived metabolites; monounsaturated fatty acids derived metabolites; amino acids, peptides, and analogues derived metabolites; D-Amino acid metabolism derived metabolites; and phenylalanine metabolism derived metabolites. Metabolites that were found to be increased in the HFpEF group included: purine metabolism-derived metabolites; cAMP signaling and mTOR signaling pathway-derived metabolites; ascorbate and aldarate metabolism derived metabolites; glyoxylate and dicarboxylate metabolism derived metabolites; and microbial metabolism in diverse environments derived metabolites (not shown).

[0269] Example 3: Double FMT humanized HFpEF microbiota mouse model

[0270] A scheme describing the experimental flow of the double FMT humanized HFpEF microbiota mouse model is provided in Fig. 4. Six groups of mice (n = 8 / group) were treated with L-NAME only until week 7 and fed with HFD throughout the experiment. Mice were administered with a first antibiotic cocktail (Abx) for 2 weeks (weeks 8 and 9). A first FMT from obese-HFpEF donors, obese-only donors, or control donors was performed at week 10 to generate humanized mice. The humanized mice were administered with a second antibiotic cocktail (Abx) for 2 weeks (weeks 13 and 14), and a second FMT (from obese-HFpEF donors, obese-only donors, or control donors) was performed on the humanized mice groups at week 15, generating double FMT groups. Echocardiology measurements were taken before each FMT and one month after each FMT, and showed that the diastolic function was significantly improved with a subsequent FMT from healthy individuals compared to mice who received additional FMT from HFpEF patients. In contrast, mice those who received the first FMT from healthy subjects followed by FMT from HFpEF patients showed worsening diastolic function (Figs. 5B-5G). As shown in Figs. 5F-5G, several metabolites were found to be increased, namely, anserine (a dipeptide comprising beta-alanine and 3-methyl-L-histidine subunits), Lastar A (2,2,6,6-Tetramethyl-4-piperidinol), N-acetylaspartate, cis-aconitic acid, and mesaconic acid; and L-iditol was found to be decreased in mice after the second antibiotic treatment following FMT from HFpEF. The improvement of symptoms following treatment mice having human FMT from HFpEF patients (humanized model for HFpEF patients) with FMT from healthy individuals provides basis for treating HFpEF patients with FMT from healthy individuals.

[0271] Example 4: Rectal treatment of HFpEF by administering bacterial populations

[0272] The HFpEF double-hit humanized mouse model is treated by oral or rectal administration of a therapeutically effective amount of isolated gut-associated bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

[0273] Diastolic and systolic parameters such as e'Wave, E / e’ ratio, left ventricle (LV) global longitudinal strain (GLS), and LV Ej ection fraction (EF) are tested in the treated mice and expected to be improved.

[0274] Example 5: Oral treatment of HFpEF by administration of antibiotics

[0275] The HFpEF double-hit humanized mouse is treated by oral or rectal administration of an antibiotic agent capable of reducing a level of Alcaligenaceae genus Sutterella and / or a Lachnospiraceae genus (possibly a genus other than Blautia or Clostridium).

[0276] Diastolic and systolic parameters such as e'Wave, E / e’ ratio, left ventricle (LV) global longitudinal strain (GLS), and LV Ejection fraction (EF) are tested in the treated mice and expected to be improved.

[0277] Example 6: Oral treatment of HFpEF by administration of agents

[0278] The HFpEF double-hit humanized mouse is treated by oral or rectal administration of at least one metabolite selected from 2-Butenoic acid, N-acetylmethionine, cis-4-Hydroxy-D-proline, and phenylacetylglycine, and / or at least one agent that blocks production or activity of a metabolite selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

[0279] Diastolic and systolic parameters such as e'Wave, E / e’ ratio, left ventricle (LV) global longitudinal strain (GLS), and LV Ejection fraction (EF) are tested in the treated mice and expected to be improved.

Claims

CLAIMSWhat is claimed is:

1. A composition for use in a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject, the composition comprising at least one of: a. fecal material from a subject not diagnosed with HFpEF; b. a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c. at least one antibiotic agent capable of reducing the abundance or activity of gut- associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d. at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and e. at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

2. The composition for use of claim 1, wherein the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are anaerobic bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

3. The composition for use of claim 1 or 2, wherein the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

4. The composition for use of any one of claims 1-3, wherein the metabolite is selected from 2- Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

5. The composition for use of any one of claims 1-4, wherein the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

6. The composition for use of any one of claims 1-5, wherein the composition comprises a therapeutically effective amount of at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautiaor Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

7. The composition for use of any one of claims 1-6, wherein the composition comprises a therapeutically effective amount of at least 2, 3, 4, 5, 6, or 7 bacterial families selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

8. The composition for use of any one of claims 1-7, wherein the composition comprises a therapeutically effective amount of at least 2, 3, 4, 5, 6, or 7 bacterial genera selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

9. The composition for use of any one of claims 1-8, wherein the composition comprises a therapeutically effective amount of at least one bacterial species selected from Parabacteroides species distasonis and Clostridium species aldenense.

10. The composition for use of any one of claims 1-9, wherein the fecal material is a fresh fecal material.

11. The composition for use of any one of claims 1-10, wherein the fecal material has been cryopreserved.

12. The composition for use of any one of claims 1-11, wherein the fecal material comprises at least one bacterial family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellaceae.

13. The composition for use of any one of claims 1-12, wherein the fecal material comprises at least one bacterial genus selected from Bacteroidaceae genus Bacteroides, Erysipelotrichaceae genus Allobaculum, Lachnospiraceae genera Blautia or Clostridium, Odoribacteraceae genus Odoribacter, Porphyromonadaceae genus Parabacteroides, and Ruminococcaceae genus Oscillospira.

14. The composition for use of any one of claims 1-13, wherein the isolated gut-associated bacteria are isolated from fecal material.

15. The composition for use of any one of claims 1-14, wherein the isolated gut-associated bacteria are cultured in vitro.

16. The composition for use of any one of claims 1-15, wherein the composition comprises at least 2, at least 3, or 4 metabolites selected from 2-Butenoic acid, N-acetylmethionine, cis-4- Hydroxy-D-proline, and phenylacetylglycine.

17. The composition for use of any one of claims 1-16, wherein the composition comprises at least 2, 3, 4, or 5 agents that blocks production or activity of at least 2, 3, 4, or 5 metabolites selected from deoxyguanosine diphosphate (dGDP), L-Threonic acid, adenylsuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

18. The composition for use of any one of claims 1-17, wherein the composition further comprises saline, glycerol, and / or polyethylene glycol (PEG).

19. The composition for use of any one of claims 1-18, wherein the subject is obese.

20. A method of treating heart failure with preserved ejection fraction (HFpEF) in a subject in need thereof, comprising administering to the subject at least one composition comprising: a. fecal material from a subject not diagnosed with HFpEF; b. a therapeutically effective amount of isolated gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance; c. at least one antibiotic agent capable of reducing the abundance or activity of gut- associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction; d. at least one metabolite selected from an amino acid, a short-chain carboxylic acid, and derivative thereof; and / or e. at least one agent that blocks production or activity of a small molecule metabolite involved in redox regulation, nucleotide metabolism, or microbial energy-associated pathways.

21. The method of claim 20, wherein the gut-associated bacteria involved in fiber metabolism, mucosal integrity, and / or immunological balance are anaerobic bacteria from at least one family selected from Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, Odoribacteraceae, Porphyromonadaceae, Ruminococcaceae, Muribaculaceae (S24-7), and Rikenellacea.

22. The method of claim 20 or 21, wherein the gut-associated bacteria involved with inflammation, dysbiosis, or metabolic dysfunction are from a genus of the family Alcaligenaceae (e.g., Sutterella) and / or of the family Lachnospiraceae.

23. The method of any one of claims 20 -22, wherein the metabolite is selected from 2-Butenoic acid, N-acetylmethionine, cis-4-hydroxy-D-proline, and phenylacetylglycine.

24. The method of any one of claims 20 -23, wherein the small molecule metabolite is selected from deoxyguanosine diphosphate (dGDP), L-threonic acid, adenylosuccinic acid, adenosine monophosphate (AMP), and mesaconic acid.

25. The method of any one of claims 20-24, wherein the administration of the at least one composition is a rectal or an oral administration.

26. The method of any one of claims 20-25, wherein the at least one composition is more than one composition.

27. The method of any one of claims 20-26, further comprising a step of administering antibiotics prior to administering the composition and / or following administering the composition.

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