Method of treatment

Abstract

This disclosure relates generally to methods of treating fatty liver diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of extracellular vesicles derived from mammalian amniotic epithelial cells.

Description

METHOD OF TREATMENTRELATED APPLICATIONS[0001 J This application claims priority to Australian Provisional Application No. 2024901327 entitled “A Method of Treatment” filed 7 May 2024, the contents of which are incorporated herein by reference in their entirety.FIELD

[0002] The present invention relates generally to methods and compositions for treating and / or preventing fatty liver disease in a subject.BACKGROUND

[0003] Pathologies such as fatty liver disease, characterized by excessive lipid accumulation in the liver is becoming the leading cause of chronic liver disease. In particular, non-alcoholic fatty liver disease (NAFLD, also referred to as metabolic dysfunction-associated steatotic liver disease (MASLD, see Rinella et al. 2023 J Hepatol 79:1542-1556), which is associated with excessive lipid accumulation in the liver without pathogenesis of fatty liver caused by excessive alcohol consumption, is a major health issue due to its close association with the worldwide epidemics of obesity and diabetes.

[0004] NAFLD covers a spectrum of hepatic lesions in the absence of alcohol intake, ranging from pure steatosis (stage 1 of NAFLD), which may progress to stage 2 NAFLD, characterised by steatosis with hepatocellular injury and inflammation, also referred to as non-alcoholic steatohepatitis or NASH (or metabolic dysfunction-associated steatohepatitis, MASH). It is a consequence of significant accumulation of triglycerides in hepatocytes, followed by oxidative stress and proinflammatory cytokine activation that lead to damaged liver tissue and scarring (fibrosis). The third stage of NAFLD is fibrosis, when there is persistent scar tissue in the liver and blood vessels around the liver. If liver damage remains untreated, fibrosis may progress, resulting in irreversible end stage liver failure, cirrhosis, and even liver cancer (hepatocarcinoma / HCC). While the recent approval of Resmetirom for the treatment of adults with noncirrhotic MASH has had an impact on the treatment possibilities for patients, the risk of drug-induced liver toxicity and potential interactions with other drugs such as statins means that there is still an unmet need for new therapeutic strategies. Early management of NAFLD / MASLD is crucial to prevent hepatic damage and other health comorbidities. Currently, the principaltreatment for NAFLD / MASLD is lifestyle modification by diet and exercise, reduction in insulin resistance, and / or treatment of diabetes. The more advanced stages of NASH are thought to need more than lifestyle modification, and several pharmacotherapies are under development. Liver transplantation (LT) is the only treatment option at the stage of decompensated cirrhosis, but with limited access due to increasing organ scarcity. NASH has also emerged as the most rapidly growing cause of hepatocellular carcinoma (HCC) among patients listed for LT, though NAFLD-related HCC diagnosed at more advanced stages (due to less systemic surveillance) can become a contraindication to transplantation.

[0005] It is estimated that 20-30% of people in Western countries suffer from nonalcoholic fatty liver, the propensity to develop this condition seems to be related to racial- ethnic origin, which could be represented in 45% Hispanic, 33% Caucasians and 24% African Americans. It has been observed that in the presence of normoglycemia and normal or moderate body weight, NAFLD is characterized by laboratory levels and clinical data similar to those observed in diabetes and obesity. Recent studies show that increasing the amount of cholesterol in the diet results in more severe inflammation, hepatocellular injury and fibrosis. On the other hand, high carbohydrate diets have been linked to an increase in the level of insulin that contributes to raising triglyceride levels. Fructose and sucrose in particular have been implicated in the development of metabolic disorders. In addition, studies have shown that the high fructose diet is associated with the development of liver inflammation, NASH and established fibrosis. Therefore, chronic excess calories and pro-inflammatory settings are understood to be linked to liver damage, in part due to the release of mediators from Kupffer cells and hepatic stellar cells (HSC), and not by hepatocytes, endothelial cells or neutrophils.

[0006] Whilst there have been advances in the treatment of fatty liver disease, current therapies have been limited by high costs and the need for specialised facilities, such as for tissue and cell retrieval, cell revival and culture. Hence, there remains an urgent need for improved methods of treating fatty liver disease.SUMMARY

[0007] The present disclosure is predicated, at least in part, on the inventors' unexpected finding that extracellular vesicles (EVs) derived from mammalian amnion epithelial cells (AECs) have therapeutic benefit in the treatment of fatty liver disease, in particular,metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH), liver cirrhosis, hepatocarcinoma (HCC). In particular, the inventors surprisingly found that EVs derived from hAECs had a therapeutic benefit during the early stages of fatty liver disease, such as non-fibrotic stages of fatty liver disease or before the development of fibrosis.

[0008] Accordingly, in one aspect, there is provided a method for treating fatty liver disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of extracellular vesicles derived from mammalian amniotic epithelial cells.

[0009] In some embodiments, the fatty liver disease is selected from the group consisting of metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH), alcohol-associated liver disease (ALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), and viral hepatitis. In an embodiment, the subject has pre-fibrotic fatty liver disease. In an embodiment, the fatty liver disease is MASLD / NAFLD or MASH / NASH. In another embodiment, the fatty liver disease is pre-fibrotic MASLD / NAFLD or MASH / NASH. In an embodiment, the fatty liver disease is simple fatty liver or steatosis.

[0010] In some embodiments, the administration of the extracellular vesicles inhibits progression of MASLD / NAFLD to MASH I NASH, as measured by: reduced NAFLD activity score; reduced steatosis; reduced fibrosis; reduced inflammation; reduced hepatocellular injury; reduced numbers of hepatic macrophages; reduced hepatocyte ballooning degeneration; and / or reduced liver progenitor cell response. In an embodiment, administration of the extracellular vesicles reduces the development of fibrosis. In an embodiment, administration of the extracellular vesicles results in a reduction in the NAFLD activity score. In an embodiment, the administration of the extracellular vesicles reduces the level of serum alanine transaminase (ALT) and / or serum aspartate aminotransferase (AST). In an embodiment, the administration of the extracellular vesicles reduces the expression of one or more lipogenesis genes and / or HCC biomarkers in liver tissue. In an embodiment, the lipogenesis gene is SREBP1 and / or SCD- 1. In an embodiment, the HCC biomarkers are one or more biomarkersselected from the group consisting of alpha fetoprotein (AFP), alpha-L-fucosidase 1 (FUCA1), glypican 3 (GPC-3) and lymphocyte antigen 6 family member D (Ly6D).

[0011] In an embodiment, administration of the extracellular vesicles inhibits progression of MASH / NASH and reduces the risk of developing liver cirrhosis.

[0012] In another embodiment, administration of the extracellular vesicles reduces the risk of developing HCC.

[0013] In some embodiments, the mammalian amniotic epithelial cells are human amniotic epithelial cells. In another embodiment, the amniotic epithelial cells are immortalised amniotic epithelial cells.

[0014] In some embodiments, the extracellular vesicles are administered to the subject at least once a month. In some embodiments, the extracellular vesicles are administered to the subject at least once a week. In an embodiment, the extracellular vesicles are administered to the subject at least twice a week. In another embodiment, the extracellular vesicles are administered to the subject at least thrice a week. In another embodiment, the extracellular vesicles are administered to the subject at least daily.

[0015] In some embodiments, the extracellular vesicles are administered orally, parenterally. In some embodiments, the extracellular vesicles are administered orally.

[0016] Accordingly, in one aspect, there is provided extracellular vesicles derived from mammalian amniotic epithelial cells for use in the treatment of treating fatty liver disease in a subject in need thereof.

[0017] In some embodiments, the fatty liver disease is selected from the group consisting of metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / non- alcoholic steatohepatitis (MASH / NASH), alcohol-associated liver disease (ALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), and viral hepatitis. In an embodiment, the subject has pre-fibrotic fatty liver disease. In an embodiment, the fatty liver disease is MASLD / NAFLD or MASH / NASH. In another embodiment, the fatty liver disease is pre-fibrotic MASLD / NAFLD or MASH / NASH.

[0018] In some embodiments, the administration of the extracellular vesicles inhibits progression of MASLD / NAFLD to MASH / NASH, as measured by: reduced NAFLD activity score; reduced steatosis; reduced fibrosis; reduced inflammation; reducedhepatocellular injury; reduced numbers of hepatic macrophages; reduced hepatocyte ballooning degeneration; and / or reduced liver progenitor cell response. In an embodiment, administration of the extracellular vesicles reduces the development of fibrosis. In an embodiment, administration of the extracellular vesicles results in a reduction in the NAFLD activity score.

[0019] In an embodiment, administration of the extracellular vesicles inhibits progression of MASH / NASH and reduces the risk of developing liver cirrhosis.

[0020] In another embodiment, administration of the extracellular vesicles reduces the risk of developing HCC.

[0021] In some embodiments, the mammalian amniotic epithelial cells are human amniotic epithelial cells. In another embodiment, the amniotic epithelial cells are immortalised amniotic epithelial cells.

[0022] In some embodiments, the extracellular vesicles are administered to the subject at least once a month. In some embodiments, the extracellular vesicles are administered to the subject at least once a week. In an embodiment, the extracellular vesicles are administered to the subject at least twice a week. In another embodiment, the extracellular vesicles are administered to the subject at least thrice a week. In another embodiment, the extracellular vesicles are administered to the subject at least daily.

[0023] In some embodiments, the extracellular vesicles are administered orally, parenterally. In some embodiments, the extracellular vesicles are administered orally.

[0024] In another aspect, there is provided a use of extracellular vesicles derived from mammalian amniotic epithelial cells in the manufacture of a medicament for the treatment of fatty liver disease.

[0025] In some embodiments, the fatty liver disease is selected from the group consisting of metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / non- alcoholic steatohepatitis (MASH / NASH), alcohol-associated liver disease (ALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), and viral hepatitis. In an embodiment, the subject has pre-fibrotic fatty liver disease. In an embodiment, the fatty liver disease is MASLD / NAFLD or MASH / NASH. In another embodiment, the fatty liver disease is pre-fibrotic MASLD / NAFLD or MASH / NASH.

[0026] In some embodiments, the administration of the extracellular vesicles inhibits progression of MASLD / NAFLD to MASH / NASH, as measured by: reduced NAFLD activity score; reduced steatosis; reduced fibrosis; reduced inflammation; reduced hepatocellular injury; reduced numbers of hepatic macrophages; reduced hepatocyte ballooning degeneration; and / or reduced liver progenitor cell response. In an embodiment, administration of the extracellular vesicles reduces the development of fibrosis. In an embodiment, administration of the extracellular vesicles results in a reduction in the NAFLD activity score.

[0027] In an embodiment, administration of the extracellular vesicles inhibits progression of MASH / NASH and reduces the risk of developing liver cirrhosis.

[0028] In another embodiment, administration of the extracellular vesicles reduces the risk of developing HCC.

[0029] In some embodiments, the mammalian amniotic epithelial cells are human amniotic epithelial cells. In another embodiment, the amniotic epithelial cells are immortalised amniotic epithelial cells.

[0030] In another aspect disclosed herein, there is provided a human amnion epithelial cell (hAEC) deposited with the Lady Mary Fairfax CellBank Australia (CBA) on 1 May 2025 and assigned Accession No. CBA20250049, and cultures thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[0032] Figure 1 is a schematic depicting the different stages of fatty liver disease.

[0033] Figure 2 is a schematic depicting the schedule for a 12-week mouse model of NAFLD to NASH. Fatty liver disease was induced in C57BL / 6 I male mice using a western ‘fast food’ diet and weekly low-dose CCL4 injections (e.g. 0.32pg CCL per gram body weight). Mice received either hAEC-EVs twice or thrice weekly via oral gavage or Obeticholic acid thrice weekly via oral gavage from week 6 to 12.

[0034] Figure 3 is a schematic depicting the schedule for a 24-week mouse model of NASH to HCC. Fatty liver disease was induced in C57BL / 6 J male mice using a western ‘fast food’ diet and weekly low-dose CCL4 injections (e.g. 0.32pg CCL per grambody weight). Mice received either hAEC-EVs twice or thrice weekly via oral gavage or Obeticholic acid thrice weekly via oral gavage from week 12 to 24.

[0035] Figure 4 depicts the metabolic profile of mice treated with experimental western ‘fast food’ diet and hAEC-EV for the 12 week NAFLD to NASH model. (A) Body weight change over 12 weeks, n=8. (B) Liver weight of mice in 12 week model. (C) Liver to body weight ratio.

[0036] Figure 5 depicts the metabolic profile of mice treated with experimental western ‘fast food’ diet and hAEC-EV for the 24 week NASH to HCC model, at the 18 week timepoint. (A) Body weight change over 118 weeks, n=8. (B) Liver weight of mice in 24 week model. (C) Liver to body weight ratio of mice in 18 week model.

[0037] Figure 6 depicts the metabolic profile of mice treated with experimental western ‘fast food’ diet and hAEC-EV for the 24 week NASH to HCC model, at the 24 week timepoint. (A) Body weight change over 24 weeks, n=8. (B) Liver weight of mice in 24 week model. (C) Liver to body weight ratio of mice in 24 week model.

[0038] Figure 7 depicts the effect of hAEC-EV administration on fibrosis in the 12 week NAFLD to NASH mice and 24 week NASH to HCC mice (A) shows Sirius Red staining of liver samples from the 12 week NAFLD to NASH mice (12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24W). Magnification: 20x, scale bar = 200 pm. (B) shows the quantification of the % of fibrosis area in the 12 week NAFLD to NASH mice (12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24W). *P < 0.05, **P< 0.001, ***P<0.01 ****P < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly.

[0039] Figure 8 depicts the effect of hAEC-EV administration on hepatic stellate cell activation in the 12 week NAFLD to NASH mice and 24 week NASH to HCC mice (A) shows hepatic alpha smooth muscle actin (aSMA) staining for activated hepatic stellate cells in the 12 week NAFLD to NASH mice and 24 week NASH to HCC mice. Magnification: 20*, scale bar = 200 pm. (B) shows the quantification of the % positive area per field area in the 12 week NAFLD to NASH mice (12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24W). *P < 0.05, **P< 0.001, ***P<0.01****P < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly.[0040J Figure 9 depicts the effect of hAEC-EV administration on macrophage numbers in the 12 week NAFLD to NASH mice and 24 week NASH to HCC mice (A) shows macrophages according to F4 / 80 positive staining in the 12 week NAFLD to NASH mice (12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24 W). Magnification: 20x, scale bar = 200 pm. (B) shows the quantification of the % positive area per field area in the 12 week NAFLD to NASH mice (12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24W). *P < 0.05, **P< 0.001, ***P<0.01 ****P < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly via oral gavage; EV2, fast food diet with hAEC-EV administration twice weekly via oral gavage; EV3, fast food diet with hAEC-EV administration thrice weekly via oral gavage.

[0041] Figure 10 depicts the effect of hAEC-EV administration on liver progenitor cell (LPCs) numbers in the 12 week NAFLD to NASH mice and 24 week NASH to HCC mice (A) shows LPCs according to PanCK staining in the 12 week NAFLD to NASH mice (12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24W). Magnification: 20*, scale bar = 200 pm. (B) shows the quantification of the number of LPCs per view of view in the 12 week NAFLD to NASH mice ( 12W) and 24 week NASH to HCC mice at 18 weeks (18W) and 24 weeks (24W). *P < 0.05, **P< 0.001, ***P<0.01 ****P < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly.

[0042] Figure 11 depicts the NAFLD inflammatory scores of (A) 12 week NAFLD to NASH mice and 24 week NASH to HCC mice (B) at 18 weeks and (C) at 24 weeks.

[0043] Figure 12 shows the expression levels of SREBP-1 and SCD1 at 12 weeks (A and D), 18 weeks (B and E) and 24 weeks timepoint (C and F). Data are represented as mean ± SEM. n=6-8, *P < 0.05, **P< 0.001, ***P<0.01 ****P < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly

[0044] Figure 13 depicts serum protein abundance as detected by SomaScan for (A) alanine aminotransferase (B) aspartate aminotransferase and (C) albumin for all treatment groups at 12 weeks, 18 weeks and 24 weeks. The box centre line indicates the median, box bounds represent 25th and 75th percentiles, and the whiskers are the most extreme values within 1.5x the interquartile range from the nearer quartile. Data presented as log2 transformed (RFU relative fluorescence units).

[0045] Figure 14 shows relative LGR5 expression in mouse liver tissue from control and treated groups. LGR5 expression was significantly lower in mice treated with hAEC-EVs (EV2 and EV3) and OCA at 12W (A), 18W(B) and 24W model (C). Data are represented as mean ± SEM. n-6-8, *P < 0.05, **P< 0.001, ***P<0.01 ****P < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly.

[0046] Figure 15 shows relative mRNA expression levels of known HCC biomarkers including Alpha-L-Fucosidase 1 (FUCA1), Glypican 3 (GPC-3) and Lymphocyte Antigen 6 Family Member D (Ly6D) and Alpha fetoprotein (AFP) in whole mouse liver tissue from control and treated groups. At 12 weeks, FUCA1 (A), GPC3 (B), LY6D (C) and AFP (D) were significantly reduced in mice treated with hAEC-EVs twice and thrice weekly. At 18 weeks, FUCA1 (E), GPC3 (F), LY6D (G) and AFP (H) were significantly reduced in mice treated with hAEC-EVs twice and thrice weekly. At 24 weeks, FUCA1 (I), LY6D (K) and AFP (L) were significantly reduced in mice treated with hAEC-EVs twice and thrice weekly. GPC3 (J) was significantly reduced in mice treated with hAEC- EVs thrice weekly. Data are represented as mean ± SEM. n=6-8, *P < 0.05, **P< 0.001, ***P<0.01 ****p < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly.

[0047] Figure 16 shows MASH-associated pathways identified through miRNA sequence analysis of hAEC-EVs. (A) Visualisation of upregulated miRNAs interacting with target genes (> 10 degrees). (B) Balloon plot of target genes by GO biological process. (C) KEGG pathways of upregulated miRNA target genes shown in the balloon plot. (D) Reactome pathways of upregulated miRNA target genes shown in the balloon plot. Red boxes highlight MASH-related pathways.

[0048] Figure 17 shows relative expression levels of miRNA targets TGFp, TLR4, TNFa, IFNy, IL-6 and IL-ip in mouse liver tissue at 12 weeks (A-F), 18 weeks (G-L) and 24 weeks (M-R). Data are represented as mean ± SEM. n=6-8, *P < 0.05, **P< 0.001, ***P<0.01 ****p < 0.0001. N, normal; FF, fast food diet alone; OCA, fast food diet with OCA thrice weekly; EV2, fast food diet with hAEC-EV administration twice weekly; EV3, fast food diet with hAEC-EV administration thrice weekly.DETAILED DESCRIPTION1. Definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0050] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0051] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0052] The terms "active agent" and "therapeutic agent" are used interchangeably herein and refer to agents that prevent, reduce or ameliorate at least one symptom of a disease or disorder.

[0053] As used herein, the terms "administration", "administering" and the like, refer to routes of administration include local administration and systemic administration. The routes of administration refer to, but are not limited to, orally, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, intrathoracically, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, rectally, topically, patch, bandage and implant. In an embodiment the EVs are locally injected or administered. In an embodiment the EVs are orally administered.

[0054] The terms “administration concurrently” or “administering concurrently” or “coadministering” and the like refer to the administration of a single composition containing two or more agents, or the administration of each agent as separate compositions and / or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such agents are administered as a single composition. By “simultaneously” is meant that the agents are administered at substantially the same time, and desirably together in the same formulation. By “contemporaneously” it is meant that the agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term “same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term “separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The agents may be administered in either order. The term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the agents may be administered in a regular repeating cycle.

[0055] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory,but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0056] The term a "disease" or "condition" also includes a "disorder". All such aspects are enabled within the width of the present invention.

[0057] By “effective amount”, in the context of treating a disease or condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and / or treating existing symptoms, of that condition. The effective amount will vary depending upon the age, health and physical condition of the individual to be treated and whether symptoms of disease are apparent, the taxonomic group of individuals to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject. Optimum dosages may vary depending on the relative potency in an individual subject, and can generally be estimated based on EC50 values found to be effective in in vitro and in vivo animal models. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0058] By ‘ ‘isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0059] The terms "subject", “patient” and “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, (e.g. human).

[0060] As used herein, the terms “treatment”, “treating”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect in a subject in need of treatment, including a subject who has fatty liver disease, in particular, metabolic dysfunction- associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH) , liver cirrhosis, hepatocarcinoma (HCC). The term "treatment", as used herein, includes ameliorating or preventing one or more symptoms or effects arising fromfatty liver disease. This may suitably include inhibition or slowing the progression of the disease. Reference to “treatment”, “treat” or “treating” is not intended to mean to completely reverse or prevent any or all symptoms or effects arising from fatty liver disease. For example, the subject may present with one or more symptoms or effects arising from fatty liver disease, but the number and / or severity of the symptoms or effects is reduced and / or the quality of life is improved as compared to prior to treatment. However, in some circumstances, the treatment may result in a complete response and / or reversal of the symptoms and effects arising from fatty liver disease. Treatment may suitable be defined by any one or more of: reduced NAFLD activity score; reduced steatosis; reduced fibrosis; reduced inflammation; reduced hepatocellular injury; reduced numbers of hepatic macrophages; reduced hepatocyte ballooning degeneration; and reduced liver progenitor cell response. Methods of assessing whether the treatment inhibited, ameliorated, or otherwise prevented one or more symptoms or effects arising from fatty liver disease will be known to persons skilled in the art. A complete response or partial response may suitably be assessed using any one of the disease scoring indices or active disease markers known to persons skilled in the art, illustrative examples of which are disclosed elsewhere herein.

[0061] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.2. Extracellular vesicles

[0062] Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular space and typically consists of heterogenous proteins, lipids, and nucleic acids. The composition and function of EVs can depend on the cell source.

[0063] In an embodiment, the EVs are derived from amnion epithelial cells (AECs). AECs are cells of the innermost lining of the placenta. Methods of isolating hAEC would be known to the person skilled in the art, an illustrative example of which is described in Murphy et al. 2010 Curr Protoc Stem Cell Biol Chapter 1: Unit IE 6 and WO2016197196.

[0064] In an embodiment, the EVs are derived from mammalian AECs. In an embodiment, the EVs are derived from human AECs (hAECs). In an embodiment, the EVs are derived from AECs of a pre-term placenta or from a term placenta. In some embodiments, the AECs may be maintained as an immortalized cell line. In an embodiment, the EVs are derived from an immortalized AEC cell line. In an embodiment,there is provided the EVs derived from one or more human amnion epithelial cells (hAEC) deposited with the Lady Mary Fairfax CellBank Australia (CBA) on 1 May 2025 and assigned Accession No. CBA20250049.

[0065] In an aspect, there is provided EVs derived from human AECs (hAEC-EVs) that comprise one or more of the miRNAs that target one or more genes encoding a target selected from the group consisting of CD44, FN1, IL6, EGFR, ITGB1, IFNG, TNF, IL10, CD4, EGF, CCL5, TLR4, IL1B, CXCR4, CXCL8, CXCL10, ICAM1 and CCR5. In an embodiment, the EVs derived from human AECs (hAEC-EVs) comprise, consist of, or consist essentially of miRNAs that target genes encoding CD44, FN1, IL6, EGFR, ITGB 1, IFNG, TNF, IL10, CD4, EGF, CCL5, TLR4, IL1B, CXCR4, CXCL8, CXCL10, ICAM1 and CCR5.

[0066] In an aspect, there is provided EVs derived from human AECs (hAEC-EVs) that comprise one or more miRNAs selected from the group consisting of hsa-let-7a-5p, hsa- let-7c-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa- mir-27a-3p, hsa-mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p. In an embodiment, the EVs derived from human AECs (hAEC-EVs) comprise hsa-let-7a-5p, hsa-let-7c-5p, hsa- let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa-mir-27a-3p, hsa- mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p. In an embodiment, the hAEC-EVs are enriched for one or more of the miRNAs selected from the group consisting of hsa-let- 7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa- mir-17-5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir- 26b-5p, hsa-mir-27a-3p, hsa-mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p. In an embodiment, the hAEC-EVs are enriched for hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir-182-5p, hsa-mir- 20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa-mir-27a-3p, hsa-mir-27b- 3p, hsa-mir-7-5p and hsa-mir-98-5p.

[0067] In an aspect, there is provided EVs derived from human AECs that comprise one or more of the miRNAs selected from the group listed in Table 3 herein. In an embodiment the hAEC-EVs comprise one or more of the miRNAs selected from the group consisting of hsa-miR-18a-5p; hsa-miR-452-5p; hsa-miR-429; hsa-miR-28-5p;hsa-miR-374a-5p; hsa-miR-182-5p; hsa-miR-21-5p; hsa-miR-3135b; hsa-miR-340-5p; hsa-miR-9903; hsa-miR-98-5p; hsa-miR-421; hsa-let-7f-5p; hsa-miR-6131; hsa-miR- 138-5p; hsa-miR-27a-3p; hsa-miR-641; hsa-miR-203a-3p; hsa-miR-30e-5p; hsa-miR-95- 3p; hsa-miR-873-5p; hsa-miR-203b-5p; hsa-miR-16-5p; hsa-miR-29b-3p; hsa-miR-30b- 5p; hsa-miR-454-3p; hsa-miR-26b-5p; hsa-miR-320d; hsa-miR-20a-5p; hsa-miR- 15b- 3p; hsa-miR-30d-3p; hsa-miR-19b-3p; hsa-miR-224-5p; hsa-miR-576-3p; hsa-miR- 27b- 3p; hsa-miR-26a-5p; hsa-miR-9985; hsa-miR-1290; hsa-miR-432-5p; hsa-miR-3074-5p; hsa-miR-96-5p; hsa-miR-218-5p; hsa-miR-148b-3p; hsa-miR-30e-3p; hsa-miR-4448; hsa-miR-934; hsa-miR-192-5p; hsa-miR-1304-3p; hsa-let-7c-5p; hsa-miR-30a-5p; hsa- let-7g-5p; hsa-miR-185-5p; hsa-miR-194-5p; hsa-miR-200b-3p; hsa-miR-3074-3p; hsa- miR-135b-5p; hsa-miR-15a-5p; hsa-miR-548d-5p; hsa-miR-22-5p; hsa-miR-548k; hsa- miR-495-3p; hsa-miR-3960; hsa-miR-101-3p; hsa-miR-450a-5p; hsa-miR-140-5p; hsa- miR-30a-3p; hsa-miR-10a-3p; hsa-miR-30c-5p; hsa-miR-1246; hsa-miR-200a-3p; hsa- miR-183-5p; hsa-let-7a-5p; hsa-miR-664a-5p; hsa-miR-152-3p; hsa-miR-23b-3p; hsa- miR-379-5p; hsa-miR-195-5p; hsa-miR-7-5p; hsa-miR-3529-3p; hsa-miR-134-5p; hsa- miR-132-3p; hsa-miR-9-5p; hsa-miR-340-3p; hsa-miR-186-5p; hsa-miR-374b-5p; hsa- miR-374c-3p; hsa-miR-23a-3p; hsa-miR-199a-3p; hsa-miR-199b-3p; hsa-miR-654-3p; hsa-miR-141-3p; hsa-miR-3065-5p; hsa-miR-582-5p; hsa-miR-140-3p; hsa-miR-664a- 3p; hsa-miR-32-3p; hsa-miR-381-3p; hsa-miR-151b; hsa-let-7f-2-3p; hsa-miR-10a-5p; hsa-miR-106b-5p; hsa-miR- 1255a; hsa-miR-17-5p; and hsa-miR-200c-5p.

[0068] In an embodiment, the hAEC-EVs are enriched for one or more of the miRNAs selected from the group listed in Table 3 herein. In an embodiment, the hAEC-EVs are enriched for hsa-miR- 18a-5p; hsa-miR-452-5p; hsa-miR-429; hsa-miR-28-5p; hsa-miR- 374a-5p; hsa-miR-182-5p; hsa-miR-21-5p; hsa-miR-3135b; hsa-miR-340-5p; hsa-miR- 9903; hsa-miR-98-5p; hsa-miR-421; hsa-let-7f-5p; hsa-miR-6131; hsa-miR-138-5p; hsa- miR-27a-3p; hsa-miR-641; hsa-miR-203a-3p; hsa-miR-30e-5p; hsa-miR-95-3p; hsa- miR-873-5p; hsa-miR-203b-5p; hsa-miR-16-5p; hsa-miR-29b-3p; hsa-miR-30b-5p; hsa- miR-454-3p; hsa-miR-26b-5p; hsa-miR-320d; hsa-miR-20a-5p; hsa-miR-15b-3p; hsa- miR-30d-3p; hsa-miR- 19b-3p; hsa-miR-224-5p; hsa-miR-576-3p; hsa-miR-27b-3p; hsa- miR-26a-5p; hsa-miR-9985; hsa-miR-1290; hsa-miR-432-5p; hsa-miR-3074-5p; hsa- miR-96-5p; hsa-miR-218-5p; hsa-miR-148b-3p; hsa-miR-30e-3p; hsa-miR-4448; hsa- miR-934; hsa-miR- 192-5p; hsa-miR- 1304-3p; hsa-let-7c-5p; hsa-miR-30a-5p; hsa-let- 7g-5p; hsa-miR- 185-5p; hsa-miR- 194-5p; hsa-miR-200b-3p; hsa-miR-3074-3p; hsa-miR-135b-5p; hsa-miR-15a-5p; hsa-miR-548d-5p; hsa-miR-22-5p; hsa-miR-548k; hsa- miR-495-3p; hsa-miR-3960; hsa-miR-101-3p; hsa-miR-450a-5p; hsa-miR-140-5p; hsa- miR-30a-3p; hsa-miR-10a-3p; hsa-miR-30c-5p; hsa-miR-1246; hsa-miR-200a-3p; hsa- miR-183-5p; hsa-let-7a-5p; hsa-miR-664a-5p; hsa-miR-152-3p; hsa-miR-23b-3p; hsa- miR-379-5p; hsa-miR-195-5p; hsa-miR-7-5p; hsa-miR-3529-3p; hsa-miR-134-5p; hsa- miR-132-3p; hsa-miR-9-5p; hsa-miR-340-3p; hsa-miR-186-5p; hsa-miR-374b-5p; hsa- miR-374c-3p; hsa-miR-23a-3p; hsa-miR-199a-3p; hsa-miR-199b-3p; hsa-miR-654-3p; hsa-miR-141-3p; hsa-miR-3065-5p; hsa-miR-582-5p; hsa-miR-140-3p; hsa-miR-664a- 3p; hsa-miR-32-3p; hsa-miR-381-3p; hsa-miR-151b; hsa-let-7f-2-3p; hsa-miR-10a-5p; hsa-miR-106b-5p; hsa-miR- 1255a; hsa-miR-17-5p; and hsa-miR-200c-5p.

[0069] In another aspect, there is provided an hAEC that is capable of producing EVs as described herein. In an embodiment, the hAEC is an immortalised hAEC. In an embodiment, the hAEC is capable of producing EVs that comprise miRNAs that target genes encoding CD44, FN1, IL6, EGFR, ITGB 1, IFNG, TNF, IL10, CD4, EGF, CCL5, TLR4, IL1B, CXCR4, CXCL8, CXCL10, ICAM1 and CCR5.

[0070] In an aspect, there is provided an hAEC that is capable of producing EVs that comprise one or more miRNAs selected from the group consisting of hsa-let-7a-5p, hsa- let-7c-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa- mir-27a-3p, hsa-mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p. In an embodiment, the hAEC that is capable of producing EVs that comprise hsa-let-7a-5p, hsa-let-7c-5p, hsa- let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa-mir-27a-3p, hsa- mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p. In an embodiment, the hAEC that is capable of producing EVs that are enriched for one or more of the miRNAs selected from the group consisting of hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir- 15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa-mir-27a-3p, hsa-mir-27b-3p, hsa-mir-7-5p and hsa- mir-98-5p. In an embodiment, the hAEC-EVs are enriched for hsa-let-7a-5p, hsa-let-7c- 5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17-5p, hsa-mir- 182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa-mir-27a- 3p, hsa-mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p.

[0071] In an aspect, there is provided an hAEC that is capable of producing EVs that comprise one or more of the miRNAs selected from the group listed in Table 3 herein. In an embodiment the hAEC is capable of producing EVs that comprise one or more of the miRNAs selected from the group consisting of hsa-miR-18a-5p; hsa-miR-452-5p; hsa- miR-429; hsa-miR-28-5p; hsa-miR-374a-5p; hsa-miR-182-5p; hsa-miR-21-5p; hsa-miR- 3135b; hsa-miR-340-5p; hsa-miR-9903; hsa-miR-98-5p; hsa-miR-421; hsa-let-7f-5p; hsa-miR-6131; hsa-miR-138-5p; hsa-miR-27a-3p; hsa-miR-641; hsa-miR-203a-3p; hsa- miR-30e-5p; hsa-miR-95-3p; hsa-miR-873-5p; hsa-miR-203b-5p; hsa-miR-16-5p; hsa- miR-29b-3p; hsa-miR-30b-5p; hsa-miR-454-3p; hsa-miR-26b-5p; hsa-miR-320d; hsa- miR-20a-5p; hsa-miR-15b-3p; hsa-miR-30d-3p; hsa-miR-19b-3p; hsa-miR-224-5p; hsa- miR-576-3p; hsa-miR-27b-3p; hsa-miR-26a-5p; hsa-miR-9985; hsa-miR-1290; hsa- miR-432-5p; hsa-miR-3074-5p; hsa-miR-96-5p; hsa-miR-218-5p; hsa-miR-148b-3p; hsa-miR-30e-3p; hsa-miR-4448; hsa-miR-934; hsa-miR-192-5p; hsa-miR-1304-3p; hsa- let-7c-5p; hsa-miR-30a-5p; hsa-let-7g-5p; hsa-miR-185-5p; hsa-miR-194-5p; hsa-miR- 200b-3p; hsa-miR-3074-3p; hsa-miR-135b-5p; hsa-miR-15a-5p; hsa-miR-548d-5p; hsa- miR-22-5p; hsa-miR-548k; hsa-miR-495-3p; hsa-miR-3960; hsa-miR-101-3p; hsa-miR- 450a-5p; hsa-miR-140-5p; hsa-miR-30a-3p; hsa-miR-10a-3p; hsa-miR-30c-5p; hsa-miR- 1246; hsa-miR-200a-3p; hsa-miR-183-5p; hsa-let-7a-5p; hsa-miR-664a-5p; hsa-miR- 152-3p; hsa-miR-23b-3p; hsa-miR-379-5p; hsa-miR-195-5p; hsa-miR-7-5p; hsa-miR- 3529-3p; hsa-miR-134-5p; hsa-miR-132-3p; hsa-miR-9-5p; hsa-miR-340-3p; hsa-miR- 186-5p; hsa-miR-374b-5p; hsa-miR-374c-3p; hsa-miR-23a-3p; hsa-miR-199a-3p; hsa- miR-199b-3p; hsa-miR-654-3p; hsa-miR-141-3p; hsa-miR-3065-5p; hsa-miR-582-5p; hsa-miR-140-3p; hsa-miR-664a-3p; hsa-miR-32-3p; hsa-miR-381-3p; hsa-miR-151b; hsa-let-7f-2-3p; hsa-miR-10a-5p; hsa-miR-106b-5p; hsa-miR- 1255a; hsa-miR-17-5p; and hsa-miR-200c-5p.

[0072] In an aspect, there is provided an hAEC that is capable of producing EVs that are enriched for one or more of the miRNAs selected from Table 3 herein.

[0073] In an embodiment, the hAEC is capable of producing EV s that is enriched for hsa- miR-18a-5p; hsa-miR-452-5p; hsa-miR-429; hsa-miR-28-5p; hsa-miR-374a-5p; hsa- miR-182-5p; hsa-miR-21-5p; hsa-miR-3135b; hsa-miR-340-5p; hsa-miR-9903; hsa- miR-98-5p; hsa-miR-421; hsa-let-7f-5p; hsa-miR-6131; hsa-miR-138-5p; hsa-miR-27a- 3p; hsa-miR-641; hsa-miR-203a-3p; hsa-miR-30e-5p; hsa-miR-95-3p; hsa-miR-873-5p;hsa-miR-203b-5p; hsa-miR-16-5p; hsa-miR-29b-3p; hsa-miR-30b-5p; hsa-miR-454-3p; hsa-miR-26b-5p; hsa-miR-320d; hsa-miR-20a-5p; hsa-miR-15b-3p; hsa-miR-30d-3p; hsa-miR-19b-3p; hsa-miR-224-5p; hsa-miR-576-3p; hsa-miR-27b-3p; hsa-miR-26a-5p; hsa-miR-9985; hsa-miR-1290; hsa-miR-432-5p; hsa-miR-3074-5p; hsa-miR-96-5p; hsa- miR-218-5p; hsa-miR-148b-3p; hsa-miR-30e-3p; hsa-miR-4448; hsa-miR-934; hsa- miR-192-5p; hsa-miR-1304-3p; hsa-let-7c-5p; hsa-miR-30a-5p; hsa-let-7g-5p; hsa-miR- 185-5p; hsa-miR-194-5p; hsa-miR-200b-3p; hsa-miR-3074-3p; hsa-miR-135b-5p; hsa- miR-15a-5p; hsa-miR-548d-5p; hsa-miR-22-5p; hsa-miR-548k; hsa-miR-495-3p; hsa- miR-3960; hsa-miR-101-3p; hsa-miR-450a-5p; hsa-miR-140-5p; hsa-miR-30a-3p; hsa- miR-10a-3p; hsa-miR-30c-5p; hsa-miR-1246; hsa-miR-200a-3p; hsa-miR-183-5p; hsa- let-7a-5p; hsa-miR-664a-5p; hsa-miR-152-3p; hsa-miR-23b-3p; hsa-miR-379-5p; hsa- miR-195-5p; hsa-miR-7-5p; hsa-miR-3529-3p; hsa-miR-134-5p; hsa-miR-132-3p; hsa- miR-9-5p; hsa-miR-340-3p; hsa-miR-186-5p; hsa-miR-374b-5p; hsa-miR-374c-3p; hsa- miR-23a-3p; hsa-miR-199a-3p; hsa-miR-199b-3p; hsa-miR-654-3p; hsa-miR-141-3p; hsa-miR-3065-5p; hsa-miR-582-5p; hsa-miR-140-3p; hsa-miR-664a-3p; hsa-miR-32-3p; hsa-miR-381-3p; hsa-miR-151b; hsa-let-7f-2-3p; hsa-miR-10a-5p; hsa-miR-106b-5p; hsa-miR-1255a; hsa-miR-17-5p; and hsa-miR-200c-5p.

[0074] In one aspect, there is provided a human amnion epithelial cell (hAEC) deposited with the Lady Mary Fairfax CellBank Australia (CBA) on 1 May 2025 and assigned Accession No. CBA20250049, and cultures thereof.3. Fatty Liver Disease and methods of treatment

[0075] Fatty liver disease (FLD), also known as hepatic steatosis and steatotic liver disease (SLD), is a condition where excess fat builds up in the liver.

[0076] The main subtypes of fatty liver disease are metabolic dysfunction-associated steatotic liver disease (MASLD, formerly "non-alcoholic fatty liver disease" (NAFLD), used interchangeably herein) and alcoholic liver disease (ALD). Non-alcoholic fatty liver disease (NAFLD) is considered a chronic liver disease characterized by excessive fat accumulation in the liver without another obvious cause (e.g. no excessive alcohol consumption, hepatotoxic medications, toxins, viral infections, genetic hepatic diseases). The term NAFLD has generally been used to refer to non-alcohol related hepatopathy. The term metabolic dysfunction-associated steatotic liver disease (MAFLD) refers to the presence of hepatic steatosis and the presence of metabolic dysregulation (e.g. Type 2diabetes, obesity). The terms NAFLD and MASLD are used interchangeably herein to refer to fatty liver disease characterized by excessive fat accumulation in the liver without obvious cause of excessive alcohol consumption, hepatotoxic medications, toxins, viral infections and / or genetic hepatic diseases.

[0077] There is a further category "metabolic and alcohol associated liver disease" (metALD), which describes an overlap of the two types of fatty liver diseases, in which patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and simultaneous moderate alcohol consumption (i.e. metALD differs from ALD based on the quantity of alcohol consumed). The deleterious association of alcohol and metabolic risk factors appears to synergistically increase the development of steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC), however the contribution of MASLD and alcohol to disease intake to the disease may vary.

[0078] NAFLD covers a spectrum of hepatic lesions in the absence of alcohol intake, ranging from pure steatosis (stage 1 of NAFLD), which may progress to stage 2 NAFLD, characterised by steatosis with hepatocellular injury and inflammation, also referred to as non-alcoholic steatohepatitis or NASH (or metabolic dysfunction-associated steatohepatitis, MASH). It is a consequence of significant accumulation of triglycerides in hepatocytes, followed by oxidative stress and proinflammatory cytokine activation that lead to damaged liver tissue and scarring (fibrosis). The third stage of NAFLD is fibrosis, when there is persistent scar tissue in the liver and blood vessels around the liver. If liver damage remains untreated, fibrosis may progress, resulting in irreversible end stage liver failure, cirrhosis, and even liver cancer (hepatocarcinoma / HCC).

[0079] In an aspect, there is provided a method for treating fatty liver disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of extracellular vesicles derived from mammalian amniotic epithelial cells. In an embodiment, the mammalian amniotic epithelial cells are of or from the same species as the subject to be treated.

[0080] In an embodiment, the fatty liver disease is selected from the group consisting of metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH), alcohol-associated liver disease (ALD), metabolic andalcohol associated liver disease" (metALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), and viral hepatitis.

[0081] MASLD / NAFLD can range from the more benign condition simple fatty liver or hepatic steatosis (an abnormal accumulation of lipids in liver cells) to non-alcoholic steatohepatitis (MASH I NASH), which is at the more severe end of the spectrum which presents with scarring, fibrosis and cirrhosis. At early stages of disease, liver cells exhibit a build-up of fat, although there is usually little to no inflammation or scarring. In some patients, fatty liver does not develop any further, and diet and lifestyle management, the excess fat in liver cells can be reduced. However, approximately 20% of people with simple fatty liver, will go on to develop MASH I NASH, wherein the build-up of fat in the liver cells is accompanied with inflammation with lobular inflammation and apoptosis. As inflammation and damage increases, and scar tissue develops and accumulates, this is known as fibrosis. Persistent and increasing scar tissue that replaces normal liver tissue impacts liver function, and can lead to liver cirrhosis (see Figure 1). The grading and stages of NAFLD / MAFLD in clinical settings are described for example in Brunt et al. (1999) Am J Gastroenterol. 94:2467 -74 Brown et al. (2016) Metabolism 65:1080-6 and Takahashi et al. (2014) World J Gastroenterol. 20:15539-48, the entire contents of which are incorporated herein by reference.

[0082] In an embodiment, the fatty liver disease is MASLD / NAFLD or MASH / NASH. In an embodiment, the fatty liver disease is MASLD / NAFL. In an embodiment, the fatty liver disease is MASH / NASH. In an embodiment, the subject has pre-fibrotic fatty liver disease. In an embodiment, the subject has hepatic steatosis. In an embodiment, the subject has pre-fibrotic MASLD / NAFLD or MASH / NASH. In an embodiment, the subject has pre-fibrotic MASLD / NAFLD. In another embodiment, the subject has MASH / NASH.

[0083] In an embodiment, administration of the extracellular vesicles has the effect of reducing NAFLD activity score, reducing steatosis, reducing fibrosis, reducing inflammation, reducing hepatocellular injury, reducing the numbers of hepatic macrophages, reducing hepatocyte ballooning degeneration, reduced liver progenitor response, reduced levels of serum alanine transaminase (ALT) and / or serum aspartate aminotransferase (AST), reduced expression of lipogenesis genes in liver tissue, and / or reduced expression of HCC biomarkers.

[0084] In an embodiment, administration of AEC-EVs reduces the development of liver fibrosis. In an embodiment, administration of AEC-EVs reduces inflammation. In an embodiment, administration of AEC-EVs reduces hepatocellular injury. In an embodiment, administration of AEC-EVs reduces the number of hepatic macrophages. In an embodiment, administration of AEC-EVs reduces hepatocyte ballooning degeneration. In an embodiment, administration of AEC-EVs reduces liver progenitor cell response.

[0085] In an embodiment, administration of AEC-EVs reduces expression of lipogenesis genes in liver tissue. Examples of lipogenesis genes would be known to the person skilled in the art, including SREBP1, ChREBP, Slug, Lsd-1, ACC, PNPLA3, TM6SF2, GCKR, APOB and SCD1. In an embodiment, administration of AEC-EVs reduces the expression of SREBP1 and SCD1 in liver tissue. In an embodiment, administration of AEC-EVs reduces steatosis. In an embodiment, administration of AEC-EVs reduces liver weight and / or the liver weight:body weight %.

[0086] In another embodiment, administration of AEC-EVs reduces the expression of one or more HCC biomarkers in liver tissue. In some embodiments, the HCC biomarker is selected from a group consisting of alpha fetoprotein (AFP), alpha-L-fucosidase 1 (FUCA1), glypican 3 (GPC-3) and / or lymphocyte antigen 6 family member D (Ly6D).

[0087] In another embodiment, administration of AEC-EVs reduces the level of serum alanine transaminase (ALT) and / or serum aspartate aminotransferase (AST). In another embodiment, administration of AEC-EVs reduces the level of serum alanine transaminase (ALT). In another embodiment, administration of AEC-EVs reduces the level of serum aspartate aminotransferase (AST).

[0088] In an embodiment, administration of AEC-EVs reduces the NAFLD activity score. The NAFLD Activity Score (NAS) is a tool used to assess the severity of nonalcoholic fatty liver disease (NAFLD) by grading the activity of liver inflammation and injury. This score is calculated by summing scores for (i) steatosis grade (fat accumulation), (ii) lobular inflammation, and (iii) hepatocyte ballooning (a type of liver cell swelling). The total NAFLD Activity Score (NAS) is the sum of these three individual scores, ranging from 0 to 8. A higher NAS indicates more severe NAFLD activity:(i) Steatosis Grade (refers to the amount of fat in the liver), scored as:• None (<5%): 0• Mild (5-33%): 1• Moderate (>33-66%): 2• Severe (>66%): 3(ii) Lobular inflammation (assesses the degree of inflammation in the liver):• No foci: 0• 1-2 foci per 200x field: 1• 3-4 foci per 200x field: 2• >4 foci per 200x field: 3(iii) Hepatocyte Ballooning (the degree of swelling of liver cells):• None: 0• Mild (few balloon cells): 1• Moderate (many balloon cells / prominent ballooning): 2

[0089] In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by at least 1. In some embodiments, the administration of AEC-EVs reduces the NAFLD activity score by around 1-8. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 1-7. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 1-6. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 1-5. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 1-4. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 1-3. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 1-2. In an embodiment, the administration of AEC- EVs reduces the NAFLD activity score by around 2. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 3. In an embodiment, the administration of AEC-EVs reduces the NAFLD activity score by around 4.

[0090] In some embodiments, the administration of AEC-EVs reduces, slows or otherwise inhibits the progression of fatty liver disease. In some embodiments, theadministration of AEC-EVs reduces, slows or otherwise inhibits the progression of MASH I NASH and reduces the risk of developing liver cirrhosis. In some embodiments, the administration of AEC-EVs reduces, slows or otherwise inhibits the progression of MASH / NASH and reduces the risk of developing liver HCC.

[0091] The amount of AEC-EVs to be administered to a subject may vary depending on, for example, on disease severity of the disease, the treatment goal, the route of administration, and the individual to be treated; and can be determined by methods well known to those skilled in the art. The dosage and / or frequency of administration may suitably be determined on an individual basis. In some embodiments, at least lOpg of AEC-EVs are administered to the subject. In some embodiments, at least 15pg of AEC- EVs are administered to the subject. In some embodiments, at least 20 ug of AEC-EVs are administered to the subject. In some embodiments, at least 25 pg of AEC-EVs are administered to the subject. In some embodiments, at least 30pg of AEC-EVs are administered to the subject. In some embodiments, at least 35 pg of AEC-EVs are administered to the subject. In some embodiments, at least 40pg of AEC-EVs are administered to the subject. In some embodiments, at least 45 pg of AEC-EVs are administered to the subject. In some embodiments, at least 50pg of AEC-EVs are administered to the subject. In some embodiments, at least 55 pg of AEC-EVs are administered to the subject. In some embodiments, at least 60pg of AEC-EVs are administered to the subject. In some embodiments, at least 65pg of AEC-EVs are administered to the subject. In some embodiments, at least 70pg of AEC-EVs are administered to the subject. In some embodiments, at least 75 pg of AEC-EVs are administered to the subject. In some embodiments, at least 80pg of AEC-EVs are administered to the subject. In some embodiments, at least 85pg of AEC-EVs are administered to the subject. In some embodiments, at least 90pg of AEC-EVs are administered to the subject. In some embodiments, at least 95 pg of AEC-EVs are administered to the subject. In some embodiments, at least 30pg of AEC-EVs are administered to the subject. In some embodiments, at east lOOpg of AEC-EVs are administered to the subject.

[0092] The AEC-EVs can be administrated as a single dose or as multiple doses. Where the AEC-EVs are administered as multiple doses, the doses may be administered hours, days, weeks or months apart. Suitable administration intervals may be determined bypersons skilled in the art and will typically depend on, for example, disease severity, treatment goal, route of administration, and the individual to be treated. For example, for more severe conditions, it might be desirable to administer multiple doses of the AEC- EVs 1 day apart, whereas for less severe conditions, it might be desirable to administer multiple doses of the AEC-EVs 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, etc.) days apart. The doses administered can be in the same amount of AEC-EVs each time. The doses administered can be in a varying amount of AEC-EVs each time. The multiple doses can be administered as a form of maintenance therapy. In an embodiment, the AEC-EVs are administered as a single dose. In another embodiment, the AEC-EVs are administered as multiple doses. In another embodiment, the AEC-EVs are administered as multiple doses at varying intervals of time. In an embodiment, the AEC-EVs are administered to the subject at least once a month. In an embodiment, the AEC-EVs are administered to the subject at least once a week. In an embodiment, the AEC-EVs are administered to the subject at least twice a week. In an embodiment, the AEC-EVs are administered to the subject at least thrice a week. In an embodiment, the AEC-EVs are administered to the subject at least thrice daily. In an embodiment, the AEC-EVs are administered as a daily dose. In an embodiment, the AEC-EVs are administered as a weekly dose. In an embodiment, the AEC-EVs are administered as a twice-weekly dose. In an embodiment, the AEC-EVs are administered as a thrice-weekly dose.

[0093] The AEC-EVs of the present invention can be administered in a pharmaceutical composition or pharmaceutical formulation. Embodiments of pharmaceutical compositions of the present disclosure comprise an effective amount of AEC-EVs dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that comprises cells are known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0094] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, hydrogels, biologic scaffolds, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible for use with AEC- EVs and / or EVs or cells, its use in the pharmaceutical compositions is contemplated.

[0095] The AEC-EVs will typically be formulated for administration in a pharmaceutically acceptable excipient or composition. Suitable pharmaceutically acceptable excipients and compositions will be familiar to persons skilled in the art, illustrative examples of which include phosphate buffered saline (PBS), isotonic saline, dextrose, dextran, heparin, Hartmann's solution, cell culture medium, amino acids, vitamins, sugars and / or proteins. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising albumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising plant albumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising recombinant albumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising ovalbumin albumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising lactalbumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising mammalian serum albumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising human serum albumin. In an embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising about 1-5% human serum albumin. In another embodiment, the AEC-EV s are formulated in a pharmaceutical composition comprising about 2.5 % human serum albumin. In another embodiment, the AEC-EVs are formulated in a pharmaceutical composition comprising about 2% human serum albumin.

[0096] The pharmaceutical formulations disclosed herein, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known to persons skilled in the art. Such techniques include the step of bringing into association the AEC-EVs with the pharmaceutical carrier(s) or excipient(s). TheAEC-EV-comprising pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including vaginal and rectal delivery), epidermal and transdermal, or parenteral. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injections or infusion. In one embodiment, the pharmaceutical composition comprising the AEC-EVs is locally injected. In one embodiment, the pharmaceutical composition comprising the AEC-EVs is formulated for oral administration. Pharmaceutical compositions and formulations for oral administration may include powders, liquids, solutions, emulsions, aerosols, gels, drops, sprays, tablets, buccal and sublingual tablets, chewable tablets, capsules, and films.EXAMPLESEXAMPLE 1: ISOLATION OF EXTRACELLULAR VESCILES DERIVED FROM HUMAN AMNION EPITHELIAL CELLS (hAEC-EVs)

[0097] Methods of isolating human amniotic epithelial cells (hAEC)-derived EVs will be known to the person skilled in the art, illustrative examples of which are described in WO2016197196; Tan etal. 2018 Stems Cells Transl Med 7: 180-196; and Zhu et al. 2022 Stem Cell Research & Therapy 13: 196, the entire contents of which are incorporated herein by reference.

[0098] In this study, term human amniotic epithelial cells (hAEC) were seeded in T175 flasks at 30,000 cells / cm2(other suitable confluency and seeding conditions would be known to the person skilled in the art) and cultured in 25mL of Optipro Serum Free media (2.5% CTS KnockOut SR XenoFree, 1% glutamax and 1% anti-antimycotic). The culture was maintained until 70% confluence was reached. The media was changed and after 3 days, conditioned media was collected for EV isolation.

[0099] The conditioned media was spun at 350g for 10 minutes to remove live cells and 2000g for 10 minutes to remove non-viable cells. The conditioned media was then concentrated using tangential flow filtration allowing for the removal of small proteinsand molecules (<20nm) in the media while concentrating and retaining the EVs in the poly sulfone hollow fibres (20 nm pores). Following the concentration of the EVs they were purified using size exclusion chromatography.

[0100] Bicinchoninic acid protein assay (BCA) was used to measure the protein concentrations of the size exclusion chromatography (SEC) fractions obtained after running SEC and analysed using Western blot. Following the Western blot, the BCA assay was used to measure the overall concentration and yield of EVs isolated, for example by measurement with SpectraMaxi3 plate reader (Molecular Devices, USA) at 562nm. The SEC fractions were validated through western blotting for an EV positive cytosolic marker, ALIX. lOpg of each fraction from each SEC column was run on a western blot and imaged to determine positive EV fractions.

[0101] Positive EV fractions from different donor placentae can be pooled and BCA was performed to determine overall yield and concentration.

[0102] The EV samples were further characterised using single -particle interferometric reflectance sensing (SP-IRIS) on the Exoview R100 platform (Nanoview Biosciences, USA) according to the manufacturer’s instructions. Briefly, multiplexed microarray chips for CD9, CD63, and CD81 tetraspanins was used to immunocapture the EVs. The images were then analysed to quantify the relative proportions of the EV surface markers CD9, CD63 and CD81 and to determine the size and quantity of EVs.EXAMPLE 2: NON-ALCOHOLIC FATTY LIVER DISEASE ACTIVITY SCORE (NAS) AND FIBROSIS

[0103] The NAFLD activity score (NAS) was determined by haemotoxylin (5 mins) and eosin (2 mins) staining of 4-pm thick deparaffmized liver sections. NAS was measured using an established scoring system that grades the extent of macrovesicular and microvesicular steatosis, hepatocyte ballooning and inflammatory foci. Liver fibrosis was graded using picrosirius red stained liver sections. Paraffin-embedded liver sections from all treatment groups were dewaxed, rehydrated and incubated for 90 min in Picrosirius red (Direct Red 80, 0.1% wt / vol in saturated picric acid, Sigma-Aldrich, St. Louis, MO, United States), then washed with acetic acid and water (1:200) and mounted in DPX (Sigma-Aldrich, St. Louis, MO, United States). 10 non-overlapping fields were acquired from all treatment groups from the 12-week, 18-week and 24-week study. Fibrosis area was further quantified by computer-assisted morphometry using Image J Fiji (NIH Image,Bethesda, MD, United States). A modified scoring system which included grading increasing extent of hepatocyte regeneration was also used to score the histological liver samples in order to improve quantification of liver fibrosis in the mice.

[0104] Paraffin-embedded liver sections from all treatment groups were dewaxed, rehydrated and incubated in 10 mM sodium citrate pH 6 or proteinase K for heat or enzyme mediated antigen retrieval. Endogenous peroxidase activity was blocked using 3% H2O2. Tissue sections were blocked with a universal protein blocking solution for 1 h and then incubated with primary antibodies overnight at 4 °C (Table 1 below). Tissue sections were then washed three times and incubated with secondary antibodies for 1 h. For PanCK, a-SMA (anti-smooth muscle actin antibody) and F4 / 80, antibody binding was detected using Vectastain ABC HRP kit (Vector Laboratories, Meadowbrook, QLD, Australia) followed by DAB chromogen (Dako, Mulgrave, Victoria, Australia). Sections for aFP were incubated with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Data are presented as number of cells per field of view (PanCK, a-SMA, F4 / 80) or percentage (%) positive cells per five non-overlapping fields at x 20 magnification (aFP) normalised to the number of DAPI positive cells using Image J (v 1.53c, National Institutes of Health, USA).

[0105] Data were analyzed using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA, United States). Murine studies were conducted with a minimum of 6 animals in each group. One way analysis of variance with Dunn's post-hoc test for multiple comparisons was performed. Data are presented as mean ± standard error of meanTable 1: Immunohistochemistry and immunofluorescence antigen retrieval, primary and secondary antibodiesEXAMPLE 3: MOUSE MODELS OF FATTY LIVER DISEASE PROGRESSION

[0106] Six to eight week old male C57BL / 6J mice (Monash Animal Research Platform, Monash University, Australia) were housed in groups of 4 to 5 in standard, wire mesh cages. Automatic lighting was allowed for a 12-h light / dark cycle (8 am-8 pm), and room temperature was maintained at 20 °C.

[0107] After acclimatization, mice were then subjected to two different diets. Control normal mice received standard chow with normal water. The other mice received a Western ‘fast food’ Diet (FF) comprising rodent chow containing 21% fat, 41% sucrose and 1.25% cholesterol and drinking water containing 23g / L d-fructose and 19g / L d- glucose (Tsuchida et al. 2018 J Hepatol. 69:385-395).

[0108] The mice that received the FF diet also received weekly injections of low dose CCL (0.32pg per gram bodyweight), to model fatty liver disease. Groups of mice that received the FF diet were also subjected to administration of lOmg / kg of obeticholic acid (OCA) thrice weekly via oral gavage, lOpgof hAEC-EVs twice weekly via oral gavage (EV2), or lOpg of hAEC-EVs thrice weekly via oral gavage (EV3), as set out in Table 2 below. Prior to administration, hAEC-EVs were thawed at room temperature and diluted in sterile phosphate -buffered saline (lOpg in lOOpI of PBS) and obeticholic acid diluted in 0.5% carboxymethyl cellulose.Table 2: Mouse treatment groupsEXAMPLE 4: 12 WEEK MOUSE MODEL OF NAFLD TO NASH

[0109] In the 12 week mouse model, the mice received either normal diet or the FF diet with low dose CCL for a total of 12 weeks. From week 6 to week 12, of the FF diet / low dose CCL mice, one group received lOpg of hAEC-EVs twice weekly via oral gavage (EV2), a second group received lOpg of hAEC-EVs thrice weekly via oral gavage (EV3) and the third group received lOmg / kg of obeticholic acid (OCA) thrice weekly via oral gavage (i.e. 6 week treatment of OCA / hAEC-EV intervention). All mice were culled at week 12 and blood and liver tissue were collected.EXAMPLE 5: 24 WEEK MOUSE MODEL OF NASH TO HCC

[0110] In the 24 week mouse model, the mice received either normal diet or the FF diet with low dose CCL for a total of 18 or 24 weeks (Tsuchida et al. 2018 J Hepatol. 69:385- 395). From week 12 to week 24 of the FF diet / low dose CCL mice, one group received lOpg of hAEC-EVs twice weekly via oral gavage (EV2), a second group received lOpg of hAEC-EVs thrice weekly via oral gavage (EV3) and the third group received lOmg / kg of obeticholic acid (OCA) thrice weekly via oral gavage. Mice were euthanised at week 18 (i.e. 6 week treatment of OCA / hAEC-EV intervention) or week 24 (i.e. 12 week treatment of OCA / hAEC-EV intervention) and blood and liver tissue were collected.EXAMPLE 6: THE EFFECT OF hAEC-EVs ON BODY AND LIVER WEIGHT

[0111] In the 12 week NAFLD to NASH model, as expected, the control FF mice (receiving the FF diet with low dose CCL and no further intervention) showed significantly increased liver weight and liver to body weight ratios. However, of the mice received the FF diet with low dose CCL, and OCA, EV2 or EV3 treatment commenced at week 6; there was a significant difference in liver weight and liver to body weight ratio in mice treated with lOpg of hAEC-EVs thrice weekly via oral gavage (EV3), but not in the OCA and EV2 treated mice at the 12 week timepoint (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) (Figure 4).

[0112] In the 24 week NASH model to HCC, as expected, the control FF mice (receiving the FF diet with low dose CCL and no further intervention) showed significantly increased liver weight and liver to body weight ratios at 18 week and 24 week timepoints. Treatment of OCA, EV2 or EV3 treatment that commenced at week 12 provided was no significant difference in liver weight or liver to body weight ratio in mice when comparedto control FF mice (receiving the FF diet with low dose CCU and no further intervention) at both 18 week and 24 week timepoints (Figure 5-6).

[0113] Serum alanine transaminase (ALT) and serum aspartate aminotransferase (AST) levels for FF mice were significantly higher compared to the mice on a normal diet for the 24- week timepoint and not at 12 weeks or 18 weeks. Additionally, compared to the FF mice, there was no significant difference in ALT or AST following treatment with hAEC-EVs twice or thrice a week or with OCA at 12 weeks, 18 weeks and 24 weeks (Figure 13A-B). There was no significant difference in serum albumin between the normal, FF, EV2, EV3 or OCA groups as detected by SomaScan (Figure 13C).EXAMPLE 7: THE EFFECT OF hAEC-EVs ON LIVER FIBROSIS IN FATTY LIVER DIEASE

[0114] Compared with normal mice, mice in both 12 week NAFLD to NASH mice and 24 week NASH to HCC model on the experimental western 'fast food' western diet (FF) had greater hepatic fibrosis areas at the measured timepoints. For the 12 week model: Normal vs. FF; 0.7% vs. 4.5%, p < 0.0001; 18-week model: Normal vs. FF; 0.7% vs. 3.8%, p < 0.0001; and 24-week model Normal vs. FF; 0.4% vs. 5.3%, p < 0.0001.

[0115] In the 12 week NAFLD to NASH mice, where OCA or hAEC-EVs commenced on week 6, treatment with hAEC-EVs thrice weekly (EV3) resulted in a significant reduction in hepatic fibrosis area compared with the untreated FF mice, whereas treatment with OCA and hAEC-EVs twice weekly (EV2) did not significantly reduce hepatic fibrosis area at 12 weeks, (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) (Figure 7).

[0116] In the 24 week NASH to HCC mice, where OCA or hAEC-EVs commenced on week 12, for the 18 week (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) and 24 week timepoints (i.e. after 12 weeks of OCA, EV2 or EV3 treatment), treatment with hAEC- EVs (EV2 and EV3) and Obeticholic Acid (OCA) resulted in a significant reduction in hepatic fibrosis area compared with the untreated FF mice (Figure 7).

[0117] Fibrosis area was significantly lower in mice treated with hAEC-EVs thrice a week at the 12- (EV3; 2.6%, P<0.05) (Figure 7A), 18- (EV3; 2.3%, P<0.001) (Figure 7B) and 24-week timepoints (EV3; 2.4%, P<0.0001) (Figure 7C) compared to untreated FF mice. Mice treated with hAEC-EVs twice a week and OCA thrice a week also showed areduction in fibrosis area at the 18 weeks (EV2=2.0%, OCA=2.3%, P<0.0001) and 24 weeks timepoint (EV2=2.6%, OCA=3.2%, P<0.0001).

[0118] Hepatic stellate cells are the primary producers of collagen in the liver and expression of a-SMA reflects the HSCs transformation to their collagen producing myofibroblast phenotype. Mice treated with hAEC-EVs twice weekly, thrice weekly and OCA thrice weekly had significantly lower numbers of a-SMA positive cells compared with untreated FF mice in both 12 week NAFLD to NASH mice and in the 24 week NASH to HCC mice (Figure 8).

[0119] ). At 12 weeks mice treated with hAEC-EVs twice weekly (4.0%, p<0.01), thrice weekly (3.0%, p<0.0001) and OCA thrice weekly (4.2%, p<0.01) had significantly lower numbers of a-SMA positive cells compared with untreated FF mice (6.0%) (Figure 8B). At 18 weeks, mice treated with hAEC-EVs twice weekly (3.8%, p<0.01), thrice weekly (3.2%, p<0.001) and OCA thrice weekly (3.3%, p<0.001) had significantly lower numbers of a-SMA positive cells compared with untreated FF mice (6.8%) (Figure 8C). At 24 weeks, mice treated with hAEC-EVs twice weekly (2.7%, p<0.0001), thrice weekly (2.5%, p<0.0001) and OCA thrice weekly (2.5%, p<0.0001) had significantly lower numbers of a-SMA positive cells compared to untreated FF mice (5.8%) (Figure 8D).EXAMPLE 8: EFFECT OF hAEC-EVs ON MACROPHAGE NUMBERS IN FATTY LIVER DIEASE

[0120] Hepatic macrophages play a crucial role in both the development of hepatic fibrosis and its resolution. Macrophages were detected through F4 / 80 staining.

[0121] Compared with normal mice, mice in both 12 week NAFLD to NASH mice and 24 week NASH to HCC model on the experimental western 'fast food' western diet (FF) had more hepatic macrophages (F4 / 80 positive cells) at the measured timepoints.

[0122] In the 12 week NAFLD to NASH mice, treatment with hAEC-EVs twice (EV2) and thrice weekly (EV3) resulted in a significant reduction in macrophage numbers compared with the untreated FF mice (twice weekly (3.4%, p<0.01) and thrice weekly (3.2%, p<0.001)). Treatment with OCA did not significantly reduce hepatic macrophages at 12 weeks, (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) (Figure 9).

[0123] In the 24 week NASH to HCC mice, where OCA or hAEC-EVs commenced on week 12, for the 18 week (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) and 24 weektimepoints (i.e. after 12 weeks of OCA, EV2 or EV3 treatment), treatment with hAEC- EVs (EV2 and EV3) and Obeticholic Acid (OCA) resulted in a significant reduction in hepatic macrophages compared with the untreated FF mice (Figure 9).

[0124] At 18 weeks, numbers of F4 / 80+ macrophages were significantly decreased in mice administered hAEC-EVs twice weekly (3.7%, p<0.0001) and thrice weekly (3.2%, p<0.0001) compared with untreated mice (6.8%) (Figure 9C). At 24 weeks, numbers of F4 / 80+ macrophages were significantly decreased in mice administered hAEC-EV s twice weekly (3.6%, p<0.0001) and thrice weekly (2.9%, p<0.0001) compared with untreated mice (9.5%) (Figure 9D). There was no difference in macrophage number among the OCA treated group at the 12 week timepoint; however, there was a significant reduction in F4 / 80+ macrophages mice treated with OCA at the 18 week (3.3% vs 6.8%, p<0.001) and 24 week timepoints (4.1% vs 9.5%, p<0.0001) compared to the untreated mice.EXAMPLE 9: EFFECT OF hAEC-EVs ON LPC RESPONSE IN FATTY LIVER DIEASE

[0125] Compared with normal mice, mice in both 12 week NAFLD to NASH mice and 24 week NASH to HCC model on the experimental western 'fast food' western diet (FF) had greater numbers of liver progenitor cells (LPCs), as judged by PanCK staining at the measured timepoints (Figure 10).

[0126] In the 12 week NAFLD to NASH mice, OCA, EV2 and EV3 treatment did not result in a significant reduction in LPC numbers when compared with the untreated FF mice.

[0127] In the 24 week NASH to HCC mice, where OCA or hAEC-EVs commenced on week 12, for the 18 week (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) and 24 week timepoints (i.e. after 12 weeks of OCA, EV2 or EV3 treatment), OCA, EV2 and EV3 treatment resulted in a significant reduction in LPC numbers when compared with the untreated FF mice (Figure 10).

[0128] At 18 weeks (Figure 10C), treatment with hAEC-EVs twice (42.5+3.6 vs 163.5±16.3, p<0.0001) and thrice weekly (28.5±2.5 vs 163.5±16.3, p<0.0001) and OCA thrice weekly (27.5+2.4 vs 163.5+16.3, p<0.0001) resulted in a significant reduction in LPC numbers compared with the untreated FF mice. At 24 weeks (Figure 10D) treatment with hAEC-EVs twice (34.3±8.1 vs 235.6±45.7, P<0.0001) and thrice weekly (33.0±4.5vs 235.6+45.7, P<0.0001) and OCA thrice weekly (33.9+2.1 vs 235.6+45.7, P<0.0001) resulted in a significant reduction in LPC numbers compared with the untreated FF mice.

[0129] Further, the number of LGR5+ cells was examined. LGR5 is a stem cell I multipotency marker, and while LGR5+ cells is usually not present in normal, healthy liver; LGR5+ cells appear upon liver damage, both acute and chronic.

[0130] A significant reduction in transcription levels of LGR5 (a stem cell I multipotency marker) was observed in mice treated with hAEC-EVs thrice weekly compared to the untreated FF mice at 12 weeks (2.8+0.8 vs 10.8+1.3, p<0.0001), 18 weeks (0.9+0.2 vs 6.1+1.0, p<0.0001) and 24 weeks (1.0+0.1 vs 6.8+1.7, p<0.001). A significant reduction in transcription levels of LGR5 in mice treated with hAEC-EVs twice weekly compared to the untreated FF mice at 12 weeks (3.9±1.0 vs 10.8±1.3, p<0.001) and 18 weeks (0.5+0.1 vs 6.1+1.0, p<0.0001). A significant reduction in transcription levels of LGR5 in mice treated with OCA thrice weekly compared to the untreated FF mice at 12 weeks (4.6±1.0 vs 1O.8±1.3, p<0.01), 18 weeks (1.5±0.2 vs 6.1+1.0, p<0.0001) and 24 weeks (3.1+0.5 vs 6.8+1.7, p<0.01) (Figure 14A-C).EXAMPLE 10: EFFECT OF hAEC-EVs ON NAFLD INFLAMMATORY ACTIVITY SCORES IN FATTY LIVER DISEASE

[0131] NAFLD activity score (NAS) was determined by haematoxylin and eosin staining of 4-pm thick deparaffinized liver sections. NAS was measured using an established scoring system that grades the extent of macrovesicular and microvesicular steatosis, hepatocyte ballooning and inflammatory foci, which is modified for rodent models, as described in Liang et al. PLoS One, (2014) 9(12): p. el 15922). Liver fibrosis was graded using picrosirius red stained liver sections. Paraffin- embedded liver sections from all treatment groups were dewaxed, rehydrated and incubated for 90 mins in Picrosirius red (Direct Red 80, 0.1% w / v in saturated picric acid, Sigma- Aldrich, St. Louis, MO, USA), then washed with acetic acid and water (1:200), dehydrated, cleared and mounted in DPX (Sigma-Aldrich, St. Louis, MO, USA). Ten non-overlapping fields were acquired from all treatment groups from the 12-week, 18-week and 24-week study. Fibrotic areas were further quantified by computer-assisted morphometry using Image J Fiji (NIH Image, Bethesda, MD, USA).

[0132] Histologically, compared with normal mice, mice in both 12 week NAFLD to NASH mice and 24 week NASH to HCC model on the experimental western 'fast food'western diet (FF) had significantly higher the NAFLD activity score (NAS) at the measured timepoints (Figures 11A-C).

[0133] In the 12 week NAFLD to NASH mice, treatment with hAEC-EVs thrice weekly (EV3) resulted in a significant reduction in activity score. OCA or EV2 treatment did not result in a significant reduction in NAFLD inflammatory activity score when compared with the untreated FF mice (Figure 11).

[0134] In the 24 week NASH to HCC mice model, where OCA or hAEC-EVs commenced on week 12, for the 18 week timepoint (i.e. after 6 weeks of OCA, EV2 or EV3 treatment) OCA, EV2 and EV3 treatments resulted in reduction in activity score compared with the untreated FF mice, but this reduction was only statistically significant for OCA and EV3. At the 24 week timepoint (i.e. after 12 weeks of OCA, EV2 or EV3 treatment), there was a similar trend in that OCA, EV2 and EV3 all resulted in a reduction in the NAFLD activity score, however these changes were not statistically significant.

[0135] Given the reduction in steatosis observed, the transcription levels of SREBP-1 (Sterol regulatory element-binding protein 1) and SCD1 (StearoyLCoA desaturase) at week 12, 18 and 24 in mouse liver tissue were assessed. SREBP-1 is a transcription factor gene related to glucose metabolism, lipogenesis and cholesterol production. Insulin- stimulated SREBP-lc increases glycolysis by activation of glucokinase enzyme and increases lipogenesis (conversion of carbohydrates into fatty acids). Suppression of SREBP-lc has been associated with protection against development of fatty liver (see Ponugoti et al. (2010) The Journal of Biological Chemistry. 285: 33959 33970 and Song et al. (2018) Nutrients. 10: El 383). SCD-1 is an enzyme that is involved in in the synthesis of unsaturated fatty acids. SCD-1 expression is significantly increased in liver tissue and heart in response to a high-carbohydrate diet. Diets high in high-saturated fat as well as monounsaturated-fat can also increase SCD-1 expression. In this regard, high expression levels of SCD1 has been correlated with obesity (Hulver MW et al. (2005) Cell Metabolism. 2: 251-61); whereas SCD-1 deficient mice have been shown to have reduced body adiposity, and improved resistance to diet-induced obesity and liver steatosis (Dobrzyn and Ntambi (2005) Obes Rev. 6: 169-74).

[0136] At 12 weeks, expression of SREBP-1 was significantly reduced in mice treated with hAEC-EVs thrice weekly (21.2±2.1, p<0.001), twice weekly (20.7±1.9, p<0.001) and with OCA (21.9+4.4, p<0.001) compared to untreated mice (54.39+8.8) (Figure12A). Furthermore, expression of SCD1 was significantly reduced in mice treated with hAEC-EVs thrice weekly compared to untreated mice (EV3 vs. FF; 12.9 ±1.2 vs 45.9±8.2, p<0.01) and but not in mice treated twice weekly or with OCA (Figure 12D). At 18 weeks expression of SREBP-1 (EV3 vs FF; 10.7+0.9 vs 15.8+1.6, p<0.05) and SCD-1 (EV3 vs FF; 10.5+1.1 vs 17.37+2.8, p<0.05) was significantly reduced in mice treated with hAEC-EVs thrice weekly compared to untreated mice. At this timepoint, mice treated twice a week with hAEC-EVs and OCA treated mice showed a reduction in SREBP1 and SCD-1 when compared to untreated mice, but this reduction was not statistically significant (Figure 12B & 12E). At 24 weeks, SREBP-1 and SCD-1 were lower in mice treated with hAEC-EV thrice and twice weekly, but this difference was not statistically significant (Figure 12C & F).

[0137] Lipotoxicity is a key driver of disease during the progression of MASLD to MASH and is driven by the imbalance of fatty acid intake, de novo lipogenesis, lipid export and fatty acid oxidation. When mice were treated with hAEC-EVs, during the progression of MASLD to MASH, there was a significant reduction in liver weight and NAFLD activity score which was less obvious at later stages of disease (18 and 24 weeks). At early stages of disease (i.e. 12 weeks) repeated doses of hAEC-EVs significantly reduced SREBP1 and SCD1 gene expression indicating the effect of hAEC- EVs on de novo lipogenesis and consequently reduce excess lipid accumulation during MASLD to MASH progression. However, there was no significant reduction to SREBP1 and SCD1 gene expression observed during MASH to HCC progression (i.e. 24 weeks).EXAMPLE 11: EFFECT OF HAEC-EVS ON HCC RISK FACTORS

[0138] MASH has been recognised as a leading predictive measure in HCC risk. Consequently, early detection of HCC development is crucial in improving the survival outcomes of patients with MASLD / MASH.

[0139] Transcriptional levels of known HCC biomarkers including alpha fetoprotein (AFP), alpha-L-fucosidase 1 (FUCA1), glypican 3 (GPC-3) and lymphocyte antigen 6 family member D (Ly6D) were assessed in mouse liver tissues. At 12 weeks, a 18-fold reduction of FUCA1 was observed in mice treated thrice weekly (EV3) (0.9+0.1 vs 16.8+3.9, p<0.0001), a 7.2-fold reduction in mice treated twice weekly with hAEC-EVs (EV2) (2.4+0.7 vsl6.8+3.9, p<0.0001) and a 4.6-fold reduction in mice treated with obeticholic acid (OCA) (3.6+0.5 vs 16.8+3.9, p<0.001) compared to untreated FF mice(Figure 15A). A 3.4-fold reduction of GPC3 was observed in the EV3 group (4.8+1.6 vs 16.3+1.2, p<0.0001), a 2.6-fold reduction in the EV2 group (6.1+1.0 vsl6.3+1.2, p<0.001) and no significant reduction when treated with OCA compared to untreated FF mice (Figure 15B). There was a 18-fold reduction in expression of LY6D in the EV3 group (4.4 + 1.7 vs 53.1 + 9.0, p<0.0001), a 2-fold reduction in the EV2 group (24.2 + 7.7 vs 53.1 ± 9.0, p<0.05) and a 2.4 fold reduction in mice treated with OCA (22.1 ± 5.2 vs 53.1 + 9.0, p<0.01) compared to untreated FF mice (Figure 15C). Further, there was a 3-fold reduction in expression of AFP in the EV3 group (34.92+6.5 vs 127.8+32.1, p<0.01), a 3-fold reduction in the EV2 group (42.3±7.5 vs 127.8±32.1, p<0.01) and a 2.7- fold reduction in mice treated with OCA (47.5+9.4 vs 127.8+32.1, p<0.01) compared to untreated FF mice (Figure 15D).

[0140] At 18 weeks, the transcription of FUCA1 was reduced by 3 -fold in the EV3 group (0.85 + 0.1 vs 3.1 + 0.4, p< 0.01) and 6-fold in the EV2 group (0.5 + 0.1 vs 3.1 + 0.4, p>0.001) compared to the untreated FF mice (Figure 15E). Furthermore, transcriptional levels of GPC-3 was reduced by 3.5-fold in the EV3 (1.4 + 0.1 vs 5.3 + 0.5, p<0.0001) and EV2 groups (1.4 + 0.3 vs 5.3 + 0.5, p<0.0001) but not significantly reduced in the OCA group compared to the untreated FF mice (Figure 15F). The transcription of Ly6D was reduced by 4-fold in the EV3 group (13.1 + 2.1 vs 54.2 + 9.1, p<0.0001), 3-fold in the EV2 group (18.5 + 2.8 vs 54.2 + 9.1, p<0.0001) and 3.5-fold in the OCA group (15.6 + 3.5 vs 54.2+ 9.1, p<0.0001) compared to untreated FF mice (Figure 15G). We observed a 3-fold reduction in expression of AFP in mice treated with hAEC-EVs thrice weekly (5.5 + 0.5 vs 17.1 + 3.1, p<0.001), a 2.5-fold reduction in mice treated with hAEC-EVs twice weekly(6.9 + 1.4 vs 17.1 + 1.3, p<0.01) and a 4.7-fold reduction in mice treated with OCA (3.6 ±0.9 vs 17.1 ± 1.3, p<0.0001) (Figure 15H).At 24 weeks, the transcription of FUCA1 was reduced by 3.6-fold in the EV3 group (1.3 ± 0.1 vs 4.7 ± 0.2, p< 0.01), 1.8-fold in the EV2 group (2.6 ± 0.1 vs 4.7 ± 0.2, p>0.01) compared to the untreated FF mice and no significant reduction when treated with OCA (Figure 151). Furthermore, transcriptional levels of GPC-3 were reduced by 4.8-fold in the EV3 group (1.3 ± 0.2 vs 6.3 ± 0.8, p<0.0001) compared to the untreated FF mice but no significant reduction in the EV2 and OCA group (Figure 15J. The transcription of Ly6D was reduced by 14.8-fold in the EV3 group (17.0 + 2.3 vs 252.3 + 37.1, p<0.0001), 16.8-fold in the EV2 group (15.5 ± 5.7 vs 252.3 ± 37.1, p<0.0001) and 14-fold in theOCA group (18.7 ± 2.4 vs 252.3 ± 37.1, p<0.0001) compared to untreated FF mice (Figure 15K). We observed a 10-fold reduction in expression of AFP in mice treated with hAEC- EVs thrice weekly (12.4 ± 2.0 vs 126 ± 28.3, p<0.01 ) and a 2.2-fold reduction in mice treated with hAEC-EVs twice weekly (55.2 ± 7.7 vs 126 ± 28.3, p<0.05) and no significant reduction in mice treated with OCA (Figure 15L).EXAMPLE 12: ANALYSIS OF MIRNA AND PROTEIN CARGO IN HAEC-EVS

[0141] miRNA-seq libraries were prepared from hAEC-EVs and subjected to sequencing and preliminary next-generation sequencing (NGS) data analyses.

[0142] Libraries were sequenced on MGI DNBSEQ-T7 Sequencer, PE 150 (Paired-End 150). There were 104 miRNAs identified in the EVs (used in the studies described above) as being differentially enriched, when compared to the single donor cell line from which they were derived (Table 3). To examine the key genes regulated by these 104 miRNAs, an integrated platform linking miRNA tool, miRNet (version 2.0) was used to predict miRNA-gene interactions. Literature- supported human ligand-receptor (LR) pairs were accessed from CellTalk (http: / / tcm.zju.edu.cn / celltalkdb / index.php). The LR pairs including the key genes as ligands or receptors were used in subsequent analyses. The protein-protein interactions (PPIs) were computed using STRING (https: / / string- db.org / ), and the hub genes in the PPIs were identified using CytoHubba in Cytoscape (3.8.2). The importance of the nodes was assessed using 6 network analysis methods including, Degree, Edge Percolated Component (EPC), Maximum Neighborhood Component (MNC), Maximal Clique Centrality (MCC) EcCentricity and Betweenness. The genes with the highest degree, EPC, MNC, MCC, EcCentricity and betweenness scores were identified as hub genes. The hub genes targeted by the upregulated miRNAs were visualized using the R package ggplot2 (v3.3.5). KEGG, GO enrichment and Reactome pathway analyses were applied to identify the common pathways and cellular processes involved in the regulation of hAEC-EVs by these hub genes.

[0143] Protein lysates from hAEC-EVs isolated (3 technical replicates) were prepared and subjected to liquid chromatography -tandem mass spectrometry (MS) analysis using DDA-based acquisition as described in Murphy et al. (2010), Current Protocols in Stem Cell Biology, 13: p. 1E.6.1-1E.6.25. For proteomic analysis, Xcalibur software v4.0 (Thermo Fisher Scientific, Waltham, MA, USA) was used to acquire MS data. RAW DDA-MS data was processed using MaxQuant (v 1.6.14.0) with its built-in search engineAndromeda. Tandem mass spectra were searched as a single batch against the Human database (UniProt, UP000005640, 79,684) supplemented with common contaminants. The search tolerance and fragment ion mass tolerance were set to 7 ppm and 0.5 Da, respectively, at less than 1 % false discovery rate on precursor and peptide spectrum match (PSM) levels employing a target-decoy approach at peptide and protein levels. Label free quantification (LFQ) algorithm in MaxQuant was used to obtain quantification intensity values and processed using Perseus as described in Rai et al. (2019) Proteomics 19: p. el800148. Contaminants, and reverse identification were excluded from further data analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the MASSive with identifier (MSV000096440).

[0144] Protein intensities were log2 transformed and normalized using quantile normalization. Hierarchical clustering was performed in Perseus, as described in Tyanova and Cox (2018) Methods Mol Biol 1711: p. 133-148, using Euclidian distance and average linkage clustering, with missing values imputed from normal distribution (width 0.3, downshift 1.8). Proteins were subjected to PCA and pairwise student’s t-test. Multiparameter (non-parametric) differential expression analysis (ANOVA, p<0.05) was performed in Perseus. Perseus, Graphpad Prism and Microsoft Excel were utilised for generating heatmaps and bar charts. For GO annotation protein accession IDs were submitted to g:Profiler. Functional enrichment analyses (Gene Ontology (GO), KEGGs) were performed using g:Profiler

[0036] , significance p<0.05. The intersection of proteins enriched in both hAEC cells and EVs was visualised using R package Venn (v.1.10). The resulting 932 intersecting proteins were enriched using the R package clusterProfiler (v.4.7), and the top gene ontology (GO) terms, KEGG pathways and Reactome pathways were visualized using the R package ggplot2 (v.3.5). The LR pairs, including the 932 intersecting proteins as ligands or receptors, were used in subsequent analyses. The hub genes in these LR pairs were identified using the method mentioned above, and the top hub genes (ligand or receptor genes) and their interacting genes were plotted using the R package ggplot2 (v.3.5). Each hub gene (ligand or receptor gene) and its interacting genes were enriched using the R package clusterProfiler (v.4.7).

[0145] miRNA sequencing and proteomics analyses were used to analyse hAEC-EV cargo to understand the therapeutic potential of hAEC-EV. Hub genes targeted byupregulated miRNAs included CD44, FN1, IL6, EGFR, ITGB1, IFNG, TNF, IL10, CD4, EGF, CCL5, TLR4, IL1B, CXCR4, CXCL8, CXCL10, ICAM1 and CCR5. miRNAs that played an important role in the regulatory network of these hub genes, with a degree of 10 or more, were visualised using miRNet 2.0. These miRNAs include hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-mir-15a-5p, hsa-mir-16-5p, hsa-mir-17- 5p, hsa-mir-182-5p, hsa-mir-20a-5p, hsa-mir-21-5p, hsa-mir-23a-3p, hsa-mir-26b-5p, hsa-mir-27a-3p, hsa-mir-27b-3p, hsa-mir-7-5p and hsa-mir-98-5p (Figure 16A).

[0146] KEGG, GO enrichment and Reactome pathway analyses were applied to identify the common pathways and biological processes regulated by these target genes. As shown in Figure 16B-D, based on pathway enrichment analysis the target genes of the upregulated miRNAs were enriched in MASH-associated pathways. Biological processes in which these genes are mainly involved include inflammatory response, immune response, PI3K / AKT signal transduction and IL-6 production (Figure 16B). KEGG analysis showed that these genes are involved in signal pathways including TLR signalling, TNF signalling, TGF P signalling, NOD-like receptor signalling and IL- 17 signalling (Figure 16C). Reactome analysis showed that these genes are involved in signal pathways including IL-4 and IL- 13 signalling, IL- 10 signalling and IFN y signalling (Figure 16D).

[0147] In order to validate whether the miRNA cargo regulates gene expression of target genes involved in enriched pathways, transcriptional levels of target genes including TGF , TLR4, TNF a, IFNy, IL-6 and IL-ip were measured by qPCR. At 12 weeks, there was a significant reduction in TGF , TLR4, TNF a, IFNy, IL-6 and IL- Ip expression in mice treated with OCA and hAEC-EVs twice and thrice weekly compared to untreated FF mice (Figure 17A-F). At 18 weeks, there was a significant reduction in TGFp, TLR4, TNF a, IFNy and IL-6 in mice treated with hAEC-EVs twice and thrice weekly compared to untreated FF mice. There was a significant reduction in TGFP, TNFa, IFNy, and IL-6 in mice treated with OCA compared to untreated FF mice. While levels of IL- IB were lower, no significant reduction was evident following repeated dosing with hAEC-EVs or OCA (Figure 17G-L). At 24 weeks, there was a significant reduction in TGFP, TLR4, TNFa, IFNy, IL-6 and IL-ip in mice treated with hAEC-EVs twice and thrice weekly compared to untreated FF mice. There was a significant reduction in TGFp, TNFa, IFNy,IL-6 and IL- 1 (3 in mice treated with OCA compared to untreated FF mice (Figure 17M- R).

[0148] Hub protein analysis revealed 10 cargo proteins enriched in hAEC-EVs. From the hub proteins identified, CD44, ITGB 1, ITGAV, CD9 and ITGA3 were receptor genes and VCL, THBS1, ICAM1, TLN1 and FN1 were ligand proteins. Based on pathway enrichment analysis the protein cargo was enriched in MASH-associated pathways which included focal adhesion, ECM-receptor interaction, extracellular matrix organisation, protein kinase B signal transduction and cell adhesion.

[0149] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0150] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0151] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

[0152] Table 3: miRNAs in EVs derived from the hAEC used in the studies described above

Claims

WHAT IS CLAIMED IS:

1. A method of treating fatty liver disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of extracellular vesicles derived from mammalian amniotic epithelial cells.

2. The method of claim 1, wherein the fatty liver disease is selected from the group consisting of metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH), alcohol-associated liver disease (ALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), and viral hepatitis.

3. The method of claim 1, wherein the fatty liver disease is simple fatty liver disease or steatosis.

4. The method of any one of claims 1-3, wherein the subject has pre-fibrotic fatty liver disease.

5. The method of any one of claims 1-4, wherein the fatty liver disease is MASLD / NAFLD or MASH / NASH.

6. The method of any one of claims 1-5, wherein the fatty liver disease is pre-fibrotic MASLD / NAFLD or MASH / NASH.

7. The method of any one of claims 1-6, wherein administration of the extracellular vesicles inhibits progression of MASLD / NAFLD to MASH / NASH, as measured by: a. reduced NAFLD activity score; b. reduced steatosis; c. reduced fibrosis; d. reduced inflammation; e. reduced hepatocellular injury; f. reduced numbers of hepatic macrophages; g. reduced hepatocyte ballooning degeneration; and / or h. reduced liver progenitor cell response.

8. The method of any one of claims 1-8, wherein administration of the extracellular vesicles reduces the level of serum alanine transaminase (ALT) and / or serum aspartate aminotransferase (AST).

9. The method of any one of claims 1-9, wherein administration of the extracellular vesicles reduces the expression of one or more lipogenesis genes and / or HCC biomarkers in liver tissue.

10. The method of claim 9, wherein the lipogenesis gene is SREBP1 and / or SCD-1.

11. The method of claim 9, wherein the HCC biomarkers are one or more biomarkers selected from the group consisting of alpha fetoprotein (AFP), alpha-L-fucosidase 1 (FUCA1), glypican 3 (GPC-3) and lymphocyte antigen 6 family member D (Ly6D).

12. The method of any one of claims 1-11, wherein administration of the extracellular vesicles reduces the development of liver fibrosis.

13. The method of any one of claims 1-12, wherein administration of the extracellular vesicles results in a reduction in the NAFLD activity score.

14. The method of any one of claims 1-13, wherein administration of the extracellular vesicles inhibits progression of MASH / NASH and reduces the risk of developing liver cirrhosis.

15. The method of any one of claims 1-14, wherein administration of the extracellular vesicles reduces the risk of developing HCC.

16. The method of any one of claims 1-15, wherein the mammalian amniotic epithelial cells are human amniotic epithelial cells.

17. The method of any one of claims 1-16, wherein the amniotic epithelial cells are immortalised amniotic epithelial cells.

18. The method of any one of claims 1-17, wherein the extracellular vesicles are administered to the subject at least once a month.

19. The method of any one of claims 1-18, wherein the extracellular vesicles are administered to the subject at least once a week.

20. The method of any one of claims 1-19, wherein the extracellular vesicles are administered to the subject at least twice a week.

21. The method of any one of claims 1-20, wherein the extracellular vesicles are administered to the subject at least thrice a week.

22. The method of any one of claims 1-21, wherein the extracellular vesicles are administered to the subject at least daily.

23. The method of any one of claims 1-22, wherein the extracellular vesicles are administered orally, parenterally, or intraperitoneally.

24. The method of any one of claims 1-23, wherein the extracellular vesicles are administered orally.

25. Extracellular vesicles derived from mammalian amniotic epithelial cells for use in the treatment of treating fatty liver disease in a subject in need thereof.

26. The extracellular vesicles for use of claim 25, wherein the fatty liver disease is selected from the group consisting of metabolic dysfunction-associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction- associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH), alcohol- associated liver disease (ALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), autoimmune hepatitis (AIH), and viral hepatitis.

27. The extracellular vesicles of claim 25, wherein the fatty liver disease is simple fatty liver disease or steatosis.

28. The extracellular vesicles for use of claim any one of clams 25-27, wherein the subject has pre-fibrotic fatty liver disease.

29. The extracellular vesicles for use of any one of claims 25 to 28, wherein the fatty liver disease is MASLD / NAFLD or MASH / NASH.

30. The extracellular vesicles for use of claim 29, wherein the fatty liver disease is pre- fibrotic MASLD / NAFLD or MASH / NASH.

31. The extracellular vesicles for use of any one of claims 25-30, wherein administration of extracellular vesicles inhibits progression of MASLD / NAFLD to MASH I NASH, as measured by: a. reduced NAFLD activity score; b. reduced steatosis; c. reduced fibrosis; d. reduced inflammation; e. reduced hepatocellular injury; f. reduced numbers of hepatic macrophages; g. reduced hepatocyte ballooning degeneration; and / or h. reduced liver progenitor cell response.

32. The extracellular vesicles of any one of claims 25-31, wherein administration of the extracellular vesicles reduces the level of serum alanine transaminase (ALT) and / or serum aspartate aminotransferase (AST).

33. The extracellular vesicles of any one of claims 25-32, wherein administration of the extracellular vesicles reduces the expression of one or more lipogenesis genes and / or HCC biomarkers in liver tissue.

34. The extracellular vesicles of claim 33, wherein the lipogenesis gene is SREBP1 and / or SCD-1.

35. The extracellular vesicles of claim 33, wherein the HCC biomarkers are one or more biomarkers selected from the group consisting of alpha fetoprotein (AFP), alpha-L- fucosidase 1 (FUCA1), glypican 3 (GPC-3) and lymphocyte antigen 6 family member D (Ly6D).

36. The extracellular vesicles for use of any one of claims 25-35, wherein administration of extracellular vesicles reduces the development of fibrosis.

37. The extracellular vesicles for use of any one of claims 25-36, wherein administration of extracellular vesicles results in a reduction in the NAFLD activity score.

38. The extracellular vesicles for use of any one of claims 25-37, wherein administration of extracellular vesicles inhibits progression of MASH / NASH and reduces the risk of developing liver cirrhosis.

39. The extracellular vesicles for use of any one of claims 25-38, wherein administration of extracellular vesicles reduces the risk of developing HCC.

40. The extracellular vesicles for use of any one of claims 25-39, wherein the mammalian amniotic epithelial cells are human amniotic epithelial cells.

41. The extracellular vesicles for use of any one of claims 25-40, wherein the mammalian amniotic epithelial cells are immortalised amniotic epithelial cells.

42. The extracellular vesicles for use of any one of claims 25-41, wherein the extracellular vesicles are administered to the subject at least once a month.

43. The extracellular vesicles for use of any one of claims 25-42, wherein the extracellular vesicles are administered to the subject at least once a week.

44. The extracellular vesicles for use of any one of claims 25-43, wherein the extracellular vesicles are administered to the subject at least twice a week.

45. The extracellular vesicles for use of any one of claims 25-44, wherein the extracellular vesicles are administered to the subject at least thrice a week.

46. The extracellular vesicles for use of any one of claims 25-45, wherein the extracellular vesicles are administered to the subject at least daily.

47. The extracellular vesiclesfor use of any one of claims 25-46, wherein the extracellular vesicles are administered orally, parenterally, or intraperitoneally.

48. The extracellular vesicles for use of any one of claims 25-47, wherein the extracellular vesicles are administered orally.

49. Use of extracellular vesicles derived from mammalian amniotic epithelial cells in the manufacture of a medicament for the treatment of fatty liver disease.

50. The use of claim 49, wherein the fatty liver disease is metabolic dysfunction- associated steatotic liver disease / non-alcoholic fatty liver disease (MASLD / NAFLD), metabolic dysfunction-associated steatohepatitis / nonalcoholic steatohepatitis (MASH / NASH), alcohol-associated liver disease (ALD), liver cirrhosis, hepatocarcinoma (HCC), drug induced liver injury (DILI), autoimmune hepatitis (AIH), and viral hepatitis.

51. The use of claim 49, wherein the fatty liver disease is simple fatty liver disease or steatosis52. The use of any one of claims 49-51, wherein the subject has pre-fibrotic fatty liver disease.

53. The use of any one of claims 49-52, wherein the fatty liver disease is MASLD / NAFLD or MASH / NASH.

54. The use of claim 53, wherein the fatty liver disease is pre-fibrotic MASLD / NAFLD or MASH / NASH.

55. The use of any one of claims 49-54, wherein administration of extracellular vesicles inhibits progression of MASLD / NAFLD to MASH / NASH, as measured by: a. reduced NAFLD activity score; b. reduced steatosis; c. reduced fibrosis; d. reduced inflammation; e. reduced hepatocellular injury; f. reduced numbers of hepatic macrophages; g. reduced hepatocyte ballooning degeneration; and / or h. reduced liver progenitor cell response.

56. The use of any one of claims 49-55, wherein administration of the extracellular vesicles reduces the level of serum alanine transaminase (ALT) and / or serum aspartate aminotransferase (AST).

57. The use of any one of claims 49-56, wherein administration of the extracellular vesicles reduces the expression of one or more lipogenesis genes and / or HCC biomarkers in liver tissue.

58. The use of any one of claims 49-57, wherein administration of extracellular vesicles reduces the development of fibrosis.

59. The use of any one of claims 49-58, wherein administration of extracellular vesicles results in a reduction in NAFLD activity score.

60. The use of any one of claims 49-59, wherein administration of extracellular vesicles inhibits progression of MASH / NASH and reduces the risk of developing liver cirrhosis.

61. The use of any one of claims 49-60, wherein administration of extracellular vesicles reduces the risk of developing HCC.

62. The use of any one of claims 49-61, wherein the mammalian amniotic epithelial cells are human amniotic epithelial cells.

63. The use of any one of claims 49-62, wherein the amniotic epithelial cells are immortalised amniotic epithelial cells.

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