WO2022182329A1

WO2022182329A1 - Phage therapy of gut microbiota dysbiosis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

Info

Publication number: WO2022182329A1
Application number: PCT/US2021/019194
Authority: WO
Inventors: Carl Merril; Greg Merril
Original assignee: Adaptive Phage Therapeutics Inc
Current assignee: Adaptive Phage Therapeutics Inc
Priority date: 2021-02-23
Filing date: 2021-02-23
Publication date: 2022-09-01
Anticipated expiration: 2023-08-23

Abstract

The invention relates to the field of phage therapy, and is particularly directed at providing a phage-based method and composition for treating the gut microbiota (GM) of a subject in a manner wherein one or more bacterial species of the GM may be specifically targeted and killed. The method and composition may be beneficial for treating a disease or condition associated with GM changes or gut dysbiosis such as NAFLD or NASH.

Description

PHAGE THERAPY OF GUT MICROBIOTA DYSBIOSIS, NON-ALCOHOLIC FATTY LIVER DISEASE AND NON-ALCOHOLIC STEATOHEPATITIS

BACKGROUND OF THE INVENTION

Field of the Invention

[001] The invention relates to the field of phage therapy, and is particularly directed at providing a phage-based method and composition for treating the gut microbiota (GM) of a subject in a manner wherein one or more bacterial species of the GM may be specifically targeted and killed. The method and composition may be beneficial for treating a disease or condition associated with GM changes or gut dysbiosis such as NAFLD or NASH.

Discussion of the Related Art

[002] In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. The Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

[003] Non-alcoholic fatty liver disease (NAFLD) is a very common disorder characterized by an excessive buildup of lipids (fat) in the liver (known as hepatic steatosis) due to causes other than the consumption of alcohol (Nonalcoholic Fatty Liver Disease & NASH, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), November 2016). It has been estimated that in the United States alone, up to one quarter of the population may be affected (i.e. 75 to 100 million Americans (Rich N.E. et ah, Clin Gastroenterol Hepatol i6(2):i98-2io, 2018)), and while typically people with NAFLD are asymptomatic (i.e. show no noticeable symptoms), a substantial portion will develop non-alcoholic steatohepatitis (NASH), which is a considerably more serious medical condition, characterized by hepatic inflammation and which can lead to cirrhosis, liver cancer, liver failure and cardiovascular disease (Nonalcoholic Fatty Liver Disease & NASH, NIDDK supra). As such, NAFLD is considered to be the leading cause of chronic liver disease (Younossi Z et ah, Nat Rev Gastroenterol Hepatol 10:330-344, 2013), and the second most common reason for liver transplantation in the United States. In 2016, the estimated annual economic burden to the United States associated with NAFLD was US$103 billion (Younossi Z et al, supra).

[004] The pathogenesis of NAFLD (and its progression to NASH) is not well understood, but given that risk factors include overweight, conditions such as insulin resistance, obesity, diabetes, dyslipidemia and high blood pressure, as well as genetic predisposition (Younossi Z et ah, supra; Friedman SL et ah, Nat Med 24(7):9o8-922, 2018), and dietary or environmental factors, it is considered that there is a complex "interplay" between these factors causing alterations in lipid metabolism subsequently leading to excessive lipid accumulation in hepatocytes, culminating in the development of NAFLD (Kolodziejczyk A et al, EMBO Mol Med n:e9302, 2019). In addition, in recent studies, it has been found that changes in the gut microbiota (GM) ("microbiome") may also play a role(s). That is, imbalances or "dysbiosis" in the makeup of the GM has been linked to the development of NAFLD (and NASH), possibly by affecting the bile acid (BA) pool or composition (which may provide signaling molecules to regulate lipid metabolism in the liver; Arab JP et al, Hepatology 65(i):350-302, 2017), and/or through influencing the balance between pro-inflammatory and anti inflammatory effectors which, in turn, might contribute to the development of inflammation in the liver (Kolodziejczyk A et al, supra).

[005] There are no specific medicinal treatments approved for NAFLD, so patients are instead "managed" by improving diet, encouraging physical exercise and avoiding alcohol consumption. Nevertheless, various treatments have been investigated and/or suggested, including the use of vitamin E and/or omega-3 fatty acid supplements, consumption of herbal compounds such as silymarin and curcumin (Bagherniya M et al, Pharmacol Res 130:213-240, 2018), and treatment with antiglycemic drugs, insulin sensitizers such as metformin, statins and drugs aimed at modulating bile acid dysregulation such as agonists of the Farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPARa) (Kolodziejczyk A et al, supra; Chitturi S et al, J Gastroenterol Hepatol 33(I):86-98, 2018). More recently, "microbiome therapy" intended to modulate the GM has been suggested. For instance, through the use of pre-, pro- and syn-biotics, and even fecal microbiota transplantation (FMT), it has been suggested that NAFLD might be attenuated by altering or correcting any gut dysbiosis (GM dysbiosis) contributing to the pathogenesis of the disease (reviewed by Ma J et ah, Nutrients 9:eii24, 2017). However, many of these treatments, which are intended to stimulate or supplement the GM, have shown, in the limited studies undertaken to date, highly variable results, perhaps due to a lack of specificity in their effect and the diversified gut dysbiosis observed among NAFLD patients. On the other hand, in circumstances where it may be desirable to inhibit or kill one or more bacterial species to correct gut dysbiosis, the use of antibiotic treatment has been suggested. However, such treatment holds concerns in terms of likely side-effects (e.g. long term use of antibiotics such as rifaximin) (Ma J et ah, supra), tolerance issues associated with long term use, and the lack of bacterial species specificity meaning that treatment may also result in off-target kills of bacterial species present in the gut at normal healthy levels (Rajpal DK et ah, Sci Prog 96(3):224-236, 2013).

[006] The present invention is directed at providing a method of treating the GM of a subject in a manner wherein one or more bacterial species of the GM (e.g. one or more bacterial species contributing to gut dysbiosis) may be specifically targeted and killed. As such, the method may be beneficial for treating a disease or condition associated with GM changes or gut dysbiosis such as NAFLD. Advantageously, the method makes use of bacteriophages ("phages"), which are a diverse set of non-cellular infective agents that replicate within and can kill specific bacterial hosts.

SUMMARY OF THE INVENTION

[007] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in any accompanying drawings and defined in the appended claims.

[008] The present invention relates to a method of treating the gut microbiota (GM) of a subject, comprising administering one or more phage type (e.g. one or more phage strains) to the subject to kill or inhibit growth of one or more bacterial species present in the subject's GM.

[009] By appropriately selecting the one or more phage type, the method may enable specific "editing" of the GM by, for example, specifically killing one or more bacterial species present in an overly high or abnormal level(s), thereby altering or correcting any gut dysbiosis. In turn, this may be beneficial for treating or attenuating the development of certain diseases or conditions including, for example, NAFLD and NASH.

[010] The invention also relates to a therapeutic phage composition for treating the gut microbiota (GM) of a subject, comprising one or more phage type (e.g. one or more phage strains) which can kill or inhibit growth of one or more bacterial species present in the subject's GM.

[011] Such a composition may comprise one or more phage types (e.g. one or more phage type(s) including phage obtained from phage "stocks") and, as such, the composition may comprise a phage "cocktail".

DETAILED DESCRIPTION

[012] The following definitions are provided for specific terms which are used in the following written description.

Definitions

[013] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Also, as understood by one of ordinary skill in the art, the term "phage" can be used to refer to a single phage or more than one phage.

[014] The present invention can "comprise" (open ended) or "consist essentially of' the components of the present invention. As used herein, "comprising" means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms "having" and "including" are also to be construed as open ended unless the context suggests otherwise. [015] The term "about" or "approximately" means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 fold, and more preferably within 2 fold, of a value. Unless otherwise stated, the term "about" means within an acceptable error range for the particular value, such as ± 1-20%, preferably ± 1-10% and more preferably ±1-5%. In even further embodiments, "about" should be understood to mean+/-5%.

[016] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[017] All ranges recited herein include the endpoints, including those that recite a range "between" two values.

[018] Terms such as "about," "generally," "substantially," "approximately" and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by one of ordinary skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.

[019] Where used herein, the term "and/or" when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition is described as containing characteristics A, B, and/ or C, the composition can contain A feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[020] The term "bacteriophage" or "phage", as understood by one of ordinary skill in the art, refers to a non-cellular infective agent that reproduces only in a suitable host cell, that is, a bacterial host cell.

[021] The term "phage therapy" refers to any therapy to treat a bacterial infection or a disease or condition that is caused by bacteria or which shows symptoms or disease/condition development or progress associated with the presence of bacteria, such as gut microbiota (GM) dysbiosis, and/or diseases or conditions which may otherwise be associated with GM dysbiosis (e.g. NAFLD or NASH). Phage therapy may involve the administration to a subject requiring treatment (e.g. a patient) of one or more therapeutic phage composition that can be used to infect, kill or inhibit growth of a bacterium, which comprises one or more viable phage as an antibacterial agent (e.g. a composition comprising one phage type or a phage "cocktail"). Where a phage therapy may involve the administration of more than one therapeutic phage composition, then the compositions may have a different host range (e.g. one may have a broad host range and one may have a narrow host range, and/ or one or more of the compositions may act synergistically with one another). Further, as will be readily understood by one of ordinary skill in the art, the therapeutic phage composition(s) used in a phage therapy will also typically comprise a range of inactive ingredients selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, and/or diluents. The term "pharmaceutically acceptable" is used to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. Examples of pharmaceutically acceptable excipients, carriers, buffers, and/or diluents are familiar to one of ordinary skill in the art and can be found, e.g. in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, Pa. For example, pharmaceutically acceptable excipients include, but are not limited to, wetting or emulsifying agents, pH buffering substances, binders, stabilizers, preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar alcohols, gelling or viscosity enhancing additives, flavoring agents, and colors. Pharmaceutically acceptable carriers include macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose, lipid aggregates (such as oil droplets or liposomes), and inactive vims particles. Pharmaceutically acceptable diluents include, but are not limited to, water and saline.

[022] The present invention relates to a method of treating the gut microbiota (GM) of a subject, comprising administering one or more phage type (e.g. one or more phage strains) to the subject to kill or inhibit growth of one or more bacterial species present in the subject's GM.

[023] In one embodiment, the one or more phage type has been selected for their capacity to kill or inhibit growth of one or more bacterial species present in the GM in an overly high ("over-represented") or abnormal level(s). The bacterial species may be non-pathogenic species commonly found in the GM of a subject, but which may be present in an overly high or abnormal level(s) when the subject is experiencing gut dysbiosis. Whether or not a particular bacterial species present in the GM of a subject is present in an overly high or abnormal level can be readily determined by standard techniques well known to one of ordinary skill in the art including, for example, 16S rRNA sequence profiling of fecal (stool) samples or cecal fluid samples (i.e. as obtained via an intestinal tube) as previously described in, for example, Le Roy T et al, Gut 62:1787-1794, 2013 and Stanley D et al., BMC Microbiol 15:51-61, 2015).

[024] By targeting bacterial species present in the GM of a subject in an overly high or abnormal level(s), the method of the invention may enable the alteration and correction of any gut dysbiosis.

[025] As such, the method of the invention may be beneficial for treating or attenuating the development of any disease or condition associated with gut dysbiosis. Accordingly, in some embodiments, the method of the invention may be performed on a subject with a disease or condition selected from, for example, metabolic diseases and conditions (e.g. overweight, obesity, insulin resistance and diabetes), Crohn's Disease, ulcerative colitis, inflammatory bowel disease (IBD), colon cancer, and asthma (Rajpal DK et al, supra). In other embodiments, the method of the invention maybe performed on a subject with a hepatic disease or condition such as, for example, NAFLD or NASH.

[026] As mentioned above, the one or more phage type administered to the subject may be selected for their capacity to kill or inhibit growth of one or more bacterial species, typically non-pathogenic bacterial species, present in the GM in an overly high or abnormal level(s). For example, the one or more phage type may be selected for their capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, B. intestinihominis, Lactobacillus, Staphylococcus, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

[027] In embodiments where the method of the invention is performed on a subject with NAFLD or NASH, the one or more phage type may be particularly selected for their capacity to kill or inhibit growth of at least one bacterial species selected from those that have been reported to be overly high in the GM of individuals within this patient group or, otherwise, in relevant animal models, including Lachnospiraceae bacterium, Barnesiella intestinihominis (Le Roy T et al, supra), Lactobacillus gasseri, Lactobacillus taiwanensis (Zeng H et al, J Nutr 143:627-631, 2013), Proteobacteria, Enterobacteria, Escherichia (Zhu L et al, Hepatology 57:601-609, 2013), Bacteroides (Boursier J et al, Hepatology 63:764-775, 2016), Gammaproteobacteria and Prevotella (Michail S et al, FEMS Microbiol Ecol 91:1-9, 2015).

[028] The one or more phage type may be selected from stocks of phage, which maybe held in storage in an inventory or "phage bank". Selection maybe performed, for example, through the use of empirical laboratory techniques that have been developed to screen for phage susceptibility on bacterial strains, such as by in vitro screening phage for their capacity to lyse (kill) or inhibit bacterial growth by testing individual phage types against a specific subject's bacterial isolate(s) from the gut using either liquid cultures or bacterial lawns grown on agar media. Bacterial isolates for screening may be readily obtained from fecal samples or cecal fluid samples taken from a subject according to standard techniques. For example, a small amount of a fecal sample (e.g. 0.1 g) may be diluted into a suitable medium (e.g. 9qqm1 of a standard culture broth) and aliquots (e.g. ioomΐ) plated out onto suitable agar plates for incubation. Following incubation, colonies may be carefully collected using routine techniques including scraping of the plate surface with a cell scraper and thereafter storing the collected samples (for later use) or culturing to expand the cell population. Aliquots of the collected samples may optionally be used to extract bacterial nucleic acid to enable identification of the bacterial species present in the fecal sample through, for example, amplification and sequencing of 16S rRNA genes by well known standard techniques (e.g. as described in Whelan FJ et al, Ann Am Thorac Soc 11:513-521, 2014). [029] Thus, in some embodiments, the one or more phage type administered to the subject has been selected by screening of phage for their capacity to lyse or inhibit growth of one or more bacterial species previously obtained from the subject's GM. Such embodiments maybe regarded as a personalized medicine approach.

[030] In other embodiments, the one or more phage type administered to the subject has been selected by screening of phage for their capacity to lyse or inhibit growth of one or more bacterial species previously obtained from the GM of at least one other subject such as another subject with the same or similar disease or condition to the subject being treated (e.g. where the method of the invention maybe performed on a subject with NAFLD, then one or more phage will have been selected by screening for their capacity to lyse or inhibit growth of one or more bacterial species previously obtained from at least one other subject with NAFLD). Where the one or more phage have been selected by screening for their capacity to lyse or inhibit growth of one or more bacterial species previously obtained from two or more other subjects with the same or similar disease or condition to the subject being treated, then such embodiments maybe regarded as an epidemiologically matched approach.

[031] The one or more phage type may be administered to the subject by any suitable route of administration. However, preferred routes of administration include oral and intravenous (i.u.) routes (e.g. i.v. infusion).

[032] The one or more phage type may be administered to the subject in the form of the therapeutic phage composition. Typically, the phage included in such a composition will comprise phage obtained from phage stocks. In some embodiments, the composition will comprise a phage cocktail.

[033] The one or more phage type may be administered to the subject in an effective amount; that is, an amount that is sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. Typically, an effective amount is sufficient for treating a disease or condition or otherwise to palliate, ameliorate, stabilize, reverse, slow or delay the progression of a disease or condition such as, for example, the development of NASH in a subject with NAFLD.

[034] The one or more phage type may be administered to the subject as a combination therapy involving the administration of a further active agent or preparation such as, for example: (i) one or more antibiotics, one or more bactericides, and/or one or more other therapeutic molecules such as a small molecule or biologic that has bactericidal activity; or (ii) one or more agents such as a small molecule or preparation intended to supplement or promote the growth one or more bacterial species present in the subject's GM such as, for example, a pre-, pro- or syn-biotic preparation. In one embodiment of such a combination therapy, which may be used for treating a subject with NAFLD or NASH, the one or more phage type is used in combination with the administration of a pre-, pro- or syn-biotic preparation for supplementing or promoting the growth of Bacteriodes vulgatus, Firmicutes, Clostridium and/or Lactobacillus (e.g. a probiotic preparation comprising viable B. vulgatus, Firmicutes spp., Clostridium spp. and/or Lactobacillus spp. including L. rhamnosus, L. plantarum, L. gasseri, L. johnsonii and L. reuteri) which have been reported as being deficient ("under-represented") in the GM of subjects with NAFLD, NASH or other metabolic diseases and conditions (Le Roy T et ah, supra; Rajpal DK et ah, supra) or which have otherwise been observed as being of potential benefit (Ma J et ah, supra).

[035] As will be readily apparent to one of ordinary skill in the art, where used in a combination therapy with one or more agents such as a small molecule or preparation intended to supplement or promote the growth one or more bacterial species present in the subject's GM, the one or more phage will be selected such that they do not kill or inhibit growth of the bacterial species that is to be supplemented or whose growth is intended to be promoted.

[036] Where used in a combination therapy with a further active agent or preparation, the one or more phage type can be administered in a single composition (i.e. a therapeutic phage composition comprising the further active agent or preparation) or in separate compositions. If administered in separate compositions, the therapeutic phage composition and the further active agent or preparation may be administered simultaneously or sequentially in any order (e.g. within seconds or minutes (e.g. 5 to 60 mins) or even hours (e.g. 2 to 48 hours)).

[037] The method of the invention will typically be performed on a human subject. However, as will be readily apparent to one of ordinary skill in the art, the method may also be useful in veterinary contexts. As such, the subject maybe an animal subject such as, for example, a livestock animal, companion animal such as a dog or cat, or exotic animal such as a lion or elephant.

[038] The invention also relates to a therapeutic phage composition for treating the gut microbiota (GM) of a subject, comprising one or more phage type (e.g. one or more phage strains) which can kill or inhibit growth of one or more bacterial species present in the subject's GM.

[039] In one embodiment, the one or more phage type has been selected for the capacity to kill or inhibit growth of one or more bacterial species (which may be non- pathogenic species commonly found in the GM of a subject) present in the GM in an overly high or abnormal level(s). For example, the one or more phage type may have been selected for the capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, B. intestinihominis, Lactobacillus, Staphylococcus, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

[040] The therapeutic phage composition may be beneficial for treating or attenuating the development of any disease or condition associated with gut dysbiosis. In some particular embodiments, the therapeutic phage composition may be used to treat a subject with a disease or condition selected from, for example, a hepatic disease or condition such as NAFLD or NASH, other metabolic diseases and conditions, Crohn's Disease, ulcerative colitis, IBD, colon cancer, and asthma.

[041] The therapeutic phage composition may be formulated for administration to the subject by any suitable route of administration, but preferably, by oral or intravenous (i.u.) routes (e.g. i.v. infusion).

[042] The therapeutic phage composition may also be formulated to include a further active agent or preparation such as: (i) one or more antibiotics, one or more bactericides, and/ or one or more other therapeutic molecules such as a small molecule or biologic that has bactericidal activity; or (ii) one or more agents such as a small molecule or preparation intended to supplement or promote the growth one or more bacterial species present in the subject's GM such as, for example, a pre-, pro- or syn- biotic preparation.

[043] In one particular embodiment of a therapeutic phage composition for treating a subject with NAFLD or NASH, the composition comprises one or more phage type selected for the capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, Barnesiella intestinihominis, Lactobacillus, Staphylococcus, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella, and a pre-, pro- or syn-biotic preparation for supplementing or promoting growth of Bacteriodes vulgatus, Firmicutes, Clostridium and/ or Lactobacillus (e.g. a probiotic preparation comprising viable B. vulgatus, Firmicutes spp., Clostridium spp. and/or Lactobacillus spp. including L. rhamnosus, L. plantarum, L. gasseri, L.johnsonii and L. reuteri).

[044] The therapeutic phage composition may be formulated so as to provide a suitable dose of each phage type included in the composition. By way of example only, a suitable dose of the phage(s) may be in the range of lo^ to io¹·’. pfu, and more preferably 109 to 10¹² pfu. Most preferably, the therapeutic phage composition is formulated to provide a dose of each phage type present of about 10¹¹ pfu.

[045] Although the invention herein has been described with reference to embodiments, it is to be understood that these embodiments, and examples provided herein, are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications can be made to the illustrative embodiments and examples, and that other arrangements can be devised without departing from the spirit and scope of the present invention as defined by the appended claims. All patent applications, patents, literature and references cited herein are hereby incorporated by reference in their entirety.

EXAMPLE(S

[046] The invention will now be further illustrated with reference to the following example(s). It will be appreciated that what follows is by way of example only and that modifications to detail may be made while still falling within the scope of the invention.

Example 1:

[047] Prophetic case study [048] A 51 year old overweight (BMI: 28 kg/m²) male, with a history of low to moderate alcohol consumption, is presented to the clinic complaining of right upper quadrant abdominal pain. He is referred for abdominal ultrasonography which showed increased hepatic echogenicity indicating the presence of fatty liver. Risk score analysis including consideration of family history of Type 2 diabetes and biochemistry including liver function tests shows a high alanine aminotransferase (ALT) level of 75 U/L ( cf normal <50 U/L), indicating that the patient is at high risk for NAFLD and liver fibrosis. Following a 6 month period during which the patient is counseled to make significant lifestyle changes (e.g. dietary and exercise changes to achieve sustained weight loss) and no alcohol consumption, liver function tests shows that his condition had worsened considerably, with results showing a substantial increase in the ALT level (105 U/L), an increase in aspartate aminotransferase (AST) such that the AST: ALT ratio is > 0.8, and also an increase in the level of alkaline phosphatase (ALP).

[049] Following referral to a gastroenterologist, the patient is tested for any gut microbiota (GM) dysbiosis that may be contributing to his condition. Fecal samples are collected from the patient and sent to a pathology laboratory for isolation of bacterial DNA and sequence analysis of the V1-V3 region of the 16S rRNA gene using a suitable 16S rRNA sequence database. As compared to age-matched control GM makeup data (i.e. generated from normal healthy individuals), this testing indicates that the patient has a very significant over-representation of B. intestinihominis within his GM. Subsequently, the recommendation is made to use phage therapy to address the patient's GM dysbiosis.

[050] Samples of the B. intestinihominis are isolated from the fecal samples obtained and screened for phage susceptibility using a proprietary phage bank using either liquid cultures or bacterial lawn grown on agar media. Subsequently, a phage is selected for the phage therapy, propagated overnight in a liquid culture of B. intestinihominis and then formulated for oral administration in a suitable liquid carrier (1 mL) to provide a dose of the phage of 10¹¹ pfu.

[051] Following the administration of the phage, further fecal samples are taken at 3 days, 7 days and 2 weeks, and each analyzed for the relative prevalence of B. intestinihominis in the GM using 16S rRNA sequence profiling. After 2 weeks, the results indicate that B. intestinihominis is no longer present in any significant level in the patient's GM.

[052] The invention is not limited to the embodiment herein before described which may be varied in construction and detail without departing from the spirit of the invention. The entire teachings of any patents, patent applications or other publications referred to herein are incorporated by reference herein as if fully set forth herein.

Claims

CLAIMS What is Claimed

1. A method of treating the gut microbiota (GM) of a subject, comprising administering one or more phage type to the subject to kill or inhibit growth of one or more bacterial species present in the subject's GM.

2. The method of claim l, wherein the one or more phage type has been selected for the capacity to kill or inhibit growth one or more bacterial species present in the GM in an overly high or abnormal level(s).

3. The method of either claim 1 or 2, wherein the bacterial species is/are non- pathogenic species commonly found in the GM of a subject, but which may be present in an overly high or abnormal level(s) when the subject is experiencing gut dysbiosis.

4. The method of any one of the previous claims, wherein the bacterial species is/ are from the group consisting of Lachnospiraceae bacterium, B. intestinihominis, Lactobacillus, Staphylococcus, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

5. The method of any one of the previous claims, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the subject's GM.

6. The method of any one of the previous claims, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the GM of at least one other subject with a disease or condition that is the same or similar to a disease or condition being experienced by the subject being treated.

7. The method of any one of claims 2-6, wherein the treatment alters or corrects gut dysbiosis in the subject.

8. The method of any one of claims 2-7, wherein the gut dysbiosis is associated with a disease or condition in the subject selected from metabolic diseases and conditions, Crohn's Disease, ulcerative colitis, inflammatory bowel disease (IBD), colon cancer, and asthma.

9. The method of any one of the previous claims, wherein the subject has NAFLD or NASH.

10. The method of any one of the previous claims, wherein the one or more phage type has been selected for the capacity to kill or inhibit growth of at least one bacterial species selected from the group consisting of Lachnospiraceae bacterium, Barnesiella intestinihominis, Lactobacillus gasseri, Lactobacillus taiwanensis, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

11. The method of any one of the previous claims, wherein the subject has a hepatic disease or condition.

12. The method of any one of the previous claims, wherein the subject has NAFLD or NASH.

13. The method of claim 12, wherein the one or more phage type has been selected for the capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, Barnesiella intestinihominis, Lactobacillus gasseri, Lactobacillus taiwanensis, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

14. The method of any one of the previous claims, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the subject's GM.

15. The method of any one of the previous claims, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the GM of at least one other subject with NAFLD or NASH.

16. The method of any one of the previous claims, wherein the one or more phage type is administered to the subject by an oral or intravenous (i.u.) route.

17. The method of any one of the previous claims, wherein the one or more phage type is administered to the subject as a combination therapy involving the administration of a further active agent or preparation.

18. The method of claim 17, wherein the further active agent or preparation is selected from: (i) one or more antibiotics, one or more bactericides, and/ or one or more other therapeutic molecules such as a small molecule or biologic that has bactericidal activity; or (ii) one or more agents such as a small molecule or preparation intended to supplement or promote the growth one or more bacterial species present in the subject's GM.

19. The method of any one of the previous claims, wherein the subject has NAFLD or NASH and the one or more phage type is administered in combination with a pre-, pro- or syn-biotic preparation for supplementing or promoting the growth of Bacteriodes vulgatus, Firmicutes, Clostridium and/or Lactobacillus.

20.A therapeutic phage composition for treating the gut microbiota (GM) of a subject, comprising one or more phage type which can kill or inhibit growth of one or more bacterial species present in the subject's GM.

21. The composition of claim 20, wherein the one or more phage type has been selected for the capacity to kill or inhibit growth one or more bacterial species present in the GM in an overly high or abnormal level(s).

22. The composition of either claim 20 or claim 21, wherein the bacterial species is/ are non-pathogenic species commonly found in the GM of a subject, but which may be present in an overly high or abnormal level(s) when the subject is experiencing gut dysbiosis.

23. The composition of any one of claims 20-22, wherein the bacterial species is/are from the group consisting of Lachnospiraceae bacterium, B. intestinihominis, Lactobacillus, Staphylococcus, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

24. The composition of any one of claims 20-23, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the subject's GM.

25. The composition of any one of claims 20-25, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the GM of at least one other subject with a disease or condition that is the same or similar to a disease or condition being experienced by the subject being treated.

26. The composition of any one of claims 20-25, wherein the treatment alters or corrects gut dysbiosis in the subject.

27. The composition of claim 26, wherein the gut dysbiosis is associated with a disease or condition in the subject selected from metabolic diseases and conditions, Crohn's Disease, ulcerative colitis, inflammatory bowel disease (IBD), colon cancer, and asthma.

28. The composition of any one of claims 20-27, wherein the subject has NAFLD or NASH.

29. The composition of claim 28, wherein the one or more phage type has been selected for the capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, Barnesiella intestinihominis, Lactobacillus gasseri, Lactobacillus taiwanensis, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

30.The composition of any one of claims 20-29, wherein the subject has a hepatic disease or condition.

31. The composition of any one of claims 20-30, wherein the subject has NAFLD or NASH.

32. The composition of any one of claims 20-31, wherein the one or more phage type has been selected for the capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, Barnesiella intestinihominis, Lactobacillus gasseri, Lactobacillus taiwanensis, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella.

33. The composition of any one of claims 20-32, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the subject's GM.

34. The composition of any one of claims 20-33, wherein the one or more phage type has been selected by in vitro screening for the capacity to kill or inhibit growth of one or more bacterial species previously obtained from the GM of at least one other subject with NAFLD or NASH.

35. The composition of any one of claims 20-34 formulated for administration to the subject by an oral or intravenous (i.u.) route.

36. The composition of claim any one of claims 20-35 formulated to include a further active agent or preparation.

37. The composition of claim 36, wherein the further active agent or preparation is selected from: (i) one or more antibiotics, one or more bactericides, and/ or one or more other therapeutic molecules such as a small molecule or biologic that has bactericidal activity; or (ii) one or more agents such as a small molecule or preparation intended to supplement or promote the growth one or more bacterial species present in the subject's GM.

38. The composition of claim 37, wherein the further preparation is a pre-, pro- or syn-biotic preparation intended to supplement or promote the growth one or more bacterial species present in the subject's GM.

39. A therapeutic phage composition for treating a subject with NAFLD or NASH, the composition comprising one or more phage type selected for the capacity to kill or inhibit growth of at least one bacterial species from the group consisting of Lachnospiraceae bacterium, Barnesiella intestinihominis, Lactobacillus, Staphylococcus, Proteobacteria, Enterobacteria, Escherichia, Bacteroides, Gammaproteobacteria and Prevotella, and a pre-, pro- or syn-biotic preparation for supplementing or promoting the growth of Bacteriodes vulgatus, Firmicutes, Clostridium and/ or Lactobacillus.