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WO2019053235A1 - Diagnostic non invasif de maladies du foie gras non alcooliques, de la stéatohépatite non alcoolique et/ou de la fibrose hépatique - Google Patents

Diagnostic non invasif de maladies du foie gras non alcooliques, de la stéatohépatite non alcoolique et/ou de la fibrose hépatique Download PDF

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WO2019053235A1
WO2019053235A1 PCT/EP2018/074977 EP2018074977W WO2019053235A1 WO 2019053235 A1 WO2019053235 A1 WO 2019053235A1 EP 2018074977 W EP2018074977 W EP 2018074977W WO 2019053235 A1 WO2019053235 A1 WO 2019053235A1
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mir
hsa
nash
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fibrosis
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Rémy HANF
Geneviève CORDONNIER
John BROZEK
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Genfit SA
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Genfit SA
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a novel non-invasive method for the diagnosis of a nonalcoholic fatty liver disease, in particular non-alcoholic steatohepatitis, and/or liver fibrosis.
  • Non-alcoholic fatty liver disease is a silent disease defined as an accumulation of fat into the liver (steatosis) for causes other than excessive alcohol consumption.
  • NAFLD is the most common cause of elevated aminotransferases in patients referred to hepatologists. NAFLD ranges from benign simple steatosis to a morbid condition for some patients, nonalcoholic steatohepatitis (NASH), where a necro/inflammatory process drives progressive accumulation of fibrosis into the liver, ultimately leading to cirrhosis, liver failure, hepatocellular carcinoma (HCC), liver transplant and liver death. Both on epidemiological and pathophysiological standpoints, NAFLD and NASH are closely associated with obesity, metabolic syndrome and type 2 diabetes.
  • NASH is considered as a growing worldwide public health issue knowing that there is no optimal solution for diagnosis and no yet approved treatment for NASH.
  • NAFLD may be diagnosed by detecting the presence of fat accumulation into the liver using ultrasound techniques
  • NASH and NASH-associated liver fibrosis can only be diagnosed by histological examination of a liver biopsy.
  • NASH is defined by fatty acid accumulation (lipid droplets) associated with damaged hepatocytes (ballooning or necrosis of the hepatocytes) and signs of lobular inflammation.
  • lipid droplets fatty acid accumulation associated with damaged hepatocytes
  • necrosis of the hepatocytes ballooning or necrosis of the hepatocytes
  • fibrosis is not a required histological feature for diagnosis of NASH
  • presence and staging of liver fibrosis is critical for assessing the severity of the disease and the risk of evolution to cirrhosis, HCC (hepatocellular carcinoma) and liver death which is the liver- related patient death.
  • the NALFD-Activity-Score has been developed for assessing the activity of the disease.
  • the NAS is the sum of the unweighted biopsy's individual scores for steatosis (0 to 3), lobular inflammation (0 to 3) and hepatocellular ballooning (0 to 2).
  • NAS is the sum of three histological scores made from liver biopsy slices:
  • HB Ballooning degeneration score: 0: none; 1 : few; 2: many cells/prominent ballooning.
  • a patient with NASH has NAS>3 and at least 1 point in steatosis, at least 1 point in lobular inflammation and at least 1 point in hepatocyte ballooning.
  • a patient is considered as having an Active-NASH when NAS>4 with at least 1 point in steatosis, at least 1 point in inflammation and at least 1 point in hepatocyte ballooning.
  • liver biopsy is an invasive procedure that may be cumbersome, worrisome and painful for the patient and liver biopsy is associated with risks of hemorrhages and even deaths. Accordingly, because of growing NASH and liver fibrosis epidemic and because biopsy cannot be seen as a sufficiently efficient and safe procedure, there is an urgent need for new non-invasive methods for diagnosis of NAFLD, NASH and/or liver fibrosis.
  • Ultrasound and imaging techniques have been developed to diagnose NAFLD.
  • these techniques are limited by both interobserver and intraobserver variability, by cost and/or are time consuming.
  • MRI-DPFF Magnetic Resonance Imaging
  • MRI-DPFF is not routinely available and is too complicated to be used in clinical practice.
  • fibrosis stage is associated with all-cause mortality in a dose dependent manner, with increased risk apparent in patients with F2 fibrosis.
  • Ultrasound-based elastography such as Fibroscan and shear wave elastography has moderate to high accuracy in diagnosing advanced fibrosis or cirrhosis.
  • F2 fibrosis is not an advanced fibrosis stage and thus cannot be accurately detected with these techniques.
  • NASH is associated with faster fibrosis progression than NAFLD and is currently the main target for pharmacological treatment. NASH patients are more likely to develop cirrhosis and die from cardiovascular and liver-related causes, with the prognostic deteriorating as the fibrosis stage progresses (Ekstedt et al, 2015). Despite the large number of serum biomarkers, combination panels, and imaging biomarkers that have been proposed, the identification of effective, less invasive, and more affordable methods for diagnosing and monitoring NAFLD, NASH and liver fibrosis are still needed, in particular methods confirmed with an independent clinical validation panel.
  • the inventors have conducted several very fine and complete analysis of different cohorts of patients to provide novel and highly sensitive non-invasive diagnostic and monitoring methods of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and liver fibrosis.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • liver fibrosis liver fibrosis.
  • the data provided herein demonstrate that miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, and miR-4324 are potent circulating biomarkers linked to NAFLD, NASH and/or liver fibrosis.
  • the methods of the present invention allow diagnosing, monitoring and risk classifying a subject as suffering from NAFLD, NASH and/or liver fibrosis.
  • the inventors also provide a method for the diagnosis, monitoring and risk classification of subjects potentially suffering from NAFLD, NAFL, NASH and/or liver fibrosis.
  • the methods of the present invention may also allow the development of new therapeutic treatments.
  • the invention provides a method for the diagnosis of a NAFLD, NASH or liver fibrosis in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324, in a body fluid sample of said subject.
  • These methods are based on the determination of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, mpiR-422a, miR-100, and miR-4324 in a body fluid of the subject.
  • the miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, and miR-4324 microRNA implemented in the present invention may be hsa-miR-132, hsa-miR-125, hsa-miR-505, hsa-miR-365, hsa-miR-22, hsa- miR-378, hsa-miR-320, hsa-miR-885, hsa-miR-483, hsa-miR-30, hsa-miR-422a, hsa-miR- 100, and hsa-miR-4324 microRNAs, such as a microRNA selected from the group consisting of hsa-miR-132-5p
  • the level of hsa-miR-125b-5p, hsa-miR-125b-2-3p, hsa-miR-505-3p, hsa-miR-365-3p, hsa-miR378a-3p, hsa-miR320e-3p, hsa-miR-885-5p, hsa-miR-483-5p, hsa-miR-30a-5p, hsa-miR-100-5p, hsa- miR-422a-5p, and/or hsa-miR-4324-3p are determined.
  • the body fluid sample may be a sample of blood, of a blood-derived fluid (such as serum and plasma, in particular platelet-free plasma, e.g. a cell-free, citrate-derived platelet-free plasma sample), of saliva, of cerebrospinal fluid or of urine.
  • a blood-derived fluid such as serum and plasma, in particular platelet-free plasma, e.g. a cell-free, citrate-derived platelet-free plasma sample
  • saliva of cerebrospinal fluid or of urine.
  • the body fluid is plasma or serum, deprived of platelets or not.
  • the body fluid level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the subject may be compared to a reference level of the respective miRNA, miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100, or miR-4324.
  • the "reference level” denotes a predetermined standard or a level determined experimentally in a sample processed similarly from a reference subject.
  • the reference subject may be a healthy subject, a subject having NAFLD but no NASH, a subject having NASH but no active NASH, or a subject with no or minimal liver fibrosis.
  • the reference subject may also be a placebo treated patient.
  • the reference level may also be the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100, or miR-4324 determined in a similarly processed body fluid sample obtained in the past from the same subject, allowing determining the evolution of NAFLD, NAFL, NASH or liver fibrosis in the subject, in particular allowing determining the evolution of the disease activity or fibrosis, or the efficiency of the treatment of the disease, depending on the method being implemented.
  • miR-30 is hsa-miR-30a, more particularly hsa-miR-30a-5p; miR- 100 is hsa-miR-100, more particularly hsa-miR-100-5p; miR-422a is hsa-miR-422a, more particularly, hsa-miR-422a-5p; or miR-4324 is hsa-miR-4324, more particularly, hsa-miR- 4324-3p.
  • the diagnosis and/or detection of NAFLD, or the diagnosis and/or detection of a potential NAFLD, in a subject is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in healthy subjects with no hepatic steatosis.
  • the diagnosis and/or detection of NAFL, or the diagnosis and/or detection of a potential NAFL, in a subject is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in healthy subjects with no hepatic steatosis, no lobular inflammation and no hepatocyte ballooning.
  • the diagnosis and/or detection of NASH, or the diagnosis and/or detection of a potential NASH, in a subject is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in a non-NASH subject such as a healthy subject, a subject with a NAS ⁇ 3 or a subject with at least one component of NAS scored at 0.
  • a non-NASH subject such as a healthy subject, a subject with a NAS ⁇ 3 or a subject with at least one component of NAS scored at 0.
  • the diagnosis and/or detection of Active-NASH, or the diagnosis and/or detection of a potential Active-NASH, in a subject is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in a healthy subject, a subject with NAS ⁇ 4 or a subject with at least one component of NAS scored at 0.
  • diagnosis and detection of significant liver fibrosis, or of potential significant liver fibrosis, in a subject is based on the detection of an increased level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in a subject with minimal liver fibrosis.
  • the diagnosis and detection of moderate liver fibrosis, or of potential moderate liver fibrosis, in a subject is based on the detection of an increased level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in a subject with significant liver fibrosis.
  • the invention relates to a method for the classification of a subject as being potential receiver (to be treated, or TBT) or non-receiver (not to be treated, or NTBT) of a treatment for NAFLD, NASH or liver fibrosis, based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level of the respective miRNA measured in NTBT patients as defined below.
  • the invention also provides a method for the determination of NAFLD activity level, NASH activity level and/or liver fibrosis stage in a subject, based on the determination of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR- 320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample of a subject.
  • the invention also allows the clinical prognostic of fibrosis, which is the prognostic of the risk of liver fibrosis evolution to cirrhosis and other liver outcomes (such as HCC and liver-related deaths) of a NAFLD or NASH patient based on the level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 determined in a body fluid sample of a subject.
  • the invention also provides a method for monitoring the evolution of NAFLD activity level, NASH activity level and/or liver fibrosis stage in a subject, based on the evolution of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample of the subject relative to a reference level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 from one or more body fluid sample(s) collected in the same subject in the past.
  • an increase of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100, or miR-4324 indicates that the disease activity and fibrosis grow up whereas a decrease of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 indicates that the disease activity and fibrosis decline.
  • the invention further provides a method for determining the efficiency of a treatment of NAFLD, NASH or liver fibrosis in a subject based on the evolution of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 in a body fluid sample of the subject relative to a reference level of the respective miRNA from one or more body fluid sample(s) collected in the same subject in the past.
  • a stable level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 may also indicate that the treatment is efficient in stabilizing the NASH, NAFLD or liver fibrosis state of the subject, thereby decreasing the risk for the subject to evolve towards critical outcomes such as cirrhosis, HCC or liver-related deaths.
  • the invention further provides a method for predicting the response of a subject (e.g. prediction of changes in NAFLD, NASH activity and liver fibrosis stage) to a specific treatment (responder subject) based on the detection of a differential level of miR-132, miR- 125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid sample relative to a reference level measured in a non-responder subject.
  • a subject e.g. prediction of changes in NAFLD, NASH activity and liver fibrosis stage
  • FIG. 1 Volcano Plot filtering of hepatic miRNA expression differences between livers of rats fed a normal diet (CSAA) and animals fed a choline deficient amino acid diet supplemented with cholesterol (CDAA + cholesterol).
  • Figure 2 A Histological profile of treatment groups. Mean of NAS, Fibrosis stage, Activity Index, and individual histological parameters was calculated in each group of the preclinical study. Results are expressed in mean ⁇ SEM.
  • Results are expressed in mean fold change relative to CSAA control group ⁇ SEM.
  • Figure 3 Correlation between plasma levels of rno-miR-132-3p with NAS, Fibrosis stage, Activity Index, Steatosis score, Hepatocyte Ballooning score, Lobular Inflammation score in rat CDAA model of NASH. All groups were used for this correlation analysis. Results are expressed as Mean ⁇ SEM.
  • TBT Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4, F ⁇ 2
  • FIG. 5 Serum level of hsa-miR-125b-5p (Up) and hsa-miR-125b-2-3p (Down) in Not-To- Be Treated (NTBT) and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Figure 9 Correlation between serum levels of hsa-miR-125b-5p with NAS, Fibrosis stage, Activity Index, Steatosis score, Hepatocyte Ballooning score, Lobular Inflammation score in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM.
  • FIG 11 Serum level of hsa-miR-505-3p in Not-To-Be Treated (NTBT) and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • NTBT2 n 161 ,
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • Figure 15 Correlation between serum levels of hsa-miR-505-3p with NAS, Fibrosis stage, Activity Index, Steatosis score, hepatocyte ballooning score, Lobular Inflammation score in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG. 16 Serum level of hsa-miR-365-3p in Not-To-Be Treated (NTBT) and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • NTBT2 n 161 ,
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; ** , p value ⁇ 0.01 .
  • Figure 20 Correlation between serum levels of hsa-miR-365-3p with NAS, Fibrosis stage, Activity Index, Steatosis score, hepatocyte ballooning score, Lobular Inflammation score in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG. 21 Serum level of hsa-miR-22-3p in Not-To-Be Treated (NTBT) and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • NTBT2 n 161 ,
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Figure 25 Correlation between serum levels of hsa-miR-22-3p with NAS, Fibrosis stage, Activity Index, Steatosis score, hepatocyte ballooning score, Lobular Inflammation score in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG. 26 Serum level of hsa-miR-378a-3p in Not-To-Be Treated (NTBT) and To-Be- Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • Figure 29 Correlation between serum levels of hsa-miR-378a-3p with NAS, Fibrosis stage, Activity Index, Steatosis score, hepatocyte ballooning score, Lobular Inflammation score in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG. 30 Serum level of hsa-miR-320e-3p in Not-To-Be Treated (NTBT) and To-Be- Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • NTBT2 n 161 ,
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; ** , p value ⁇ 0.01 .
  • Figure 33 Correlation between serum levels of hsa-miR-320e-3p with NAS, Fibrosis stage, Activity Index, Steatosis score, hepatocyte ballooning score, Lobular Inflammation score in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG. 34 Serum level of hsa-miR-885-5p in Not-To-Be Treated (NTBT) and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • NTBT2 n 161 ,
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Figure 38 Correlation between serum levels of hsa-miR-885-5p with NAS, Fibrosis stage, activity Index, steatosis score, hepatocyte ballooning score (HB), lobular Inflammation score (LI) in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG 39 Serum level of hsa-miR-483-5p in Not-To-Be Treated (NTBT) and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • NTBT2 n 161 ,
  • Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Mann Whitney test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • Figure 42 Correlation between serum levels of hsa-miR-483-5p with NAS, Fibrosis stage, activity Index, steatosis score, hepatocyte ballooning score (HB), lobular Inflammation score (LI) in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • FIG 43 Serum level of hsa-miR-30a-5p in Healthy subjects, Not-To-Be Treated (NTBT), and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • Results are expressed as Mean ⁇ SEM.
  • Statistical analyses were conducted using one way ANOVA followed by Holm- Sidak's multiple comparisons test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Figure 44 Serum level of hsa-miR-100-5p in Healthy subjects, Not-To-Be Treated (NTBT), and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • FIG. 45 Serum level of hsa-miR-422a in Healthy subjects, Not-To-Be Treated (NTBT), and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • Results are expressed as Mean ⁇ SEM.
  • Statistical analyses were conducted using one way ANOVA followed by Holm- Sidak's multiple comparisons test: *** , p value ⁇ 0.001 ; * , p value ⁇ 0.05.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Figure 46 Serum level of hsa-miR-4324 in Healthy subjects, Not-To-Be Treated (NTBT), and To-Be-Treated (TBT) patients of GOLDEN-DIAG according to three different definitions of TBT patients: TBT1 , TBT2 and TBT7.
  • TBT1 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 1 TBT2 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F ⁇ 2 TBT7 Steatosis, lobular inflammation and hepatocyte ballooning score ⁇ 1 , NAS ⁇ 4,
  • F 1 b, 1 c, 2, 3 or 4
  • Figure 51 Correlation between serum levels of hsa-miR-30a-5p with NAS, Fibrosis stage, activity Index, steatosis score, hepatocyte ballooning score (HB), lobular Inflammation score (LI) in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001 .
  • Figure 52 Correlation between serum levels of hsa-miR-422a with NAS, Fibrosis stage, activity Index, steatosis score, hepatocyte ballooning score (HB), lobular Inflammation score (LI) in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001.
  • Figure 53 Correlation between serum levels of hsa-miR-100-5p with NAS, Fibrosis stage, activity Index, steatosis score, hepatocyte ballooning score (HB), lobular Inflammation score (LI) in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001 .
  • Figure 54 Correlation between serum levels of hsa-miR-4324 with NAS, Fibrosis stage, activity Index, steatosis score, hepatocyte ballooning score (HB), lobular Inflammation score (LI) in patients of GOLDEN-DIAG cohort at inclusion. Results are expressed as Mean ⁇ SEM. Statistical significance was calculated using Kruskal Wallis ANOVA test followed by Dunn's multiple comparison test: * , p value ⁇ 0.05; ** p value ⁇ 0.005; *** , p value ⁇ 0.001. DETAILED DESCRIPTION OF THE INVENTION
  • the inventors provide a new method for the diagnosis, monitoring and risk classification of subjects suffering or potentially suffering from NAFLD, NASH and/or liver fibrosis.
  • the present invention stems from the very fine analysis of patients' biopsies during a clinical trial, to correlate the presence or level of circulating biological markers and to classify patients as to be treated or not to be treated.
  • the present invention non- limitatively defines three classes of NASH patients to be treated. These patients are classified with respect to the scoring of NASH characteristics.
  • the experimental data provided herein surprisingly identify miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324 as a circulating biomarker for NAFLD, NASH and/or liver fibrosis from three large independent cohorts of patients, namely GOLDEN-DIAG (N 270 at inclusion) with scored liver biopsies and corresponding blood, plasma and serum samples.
  • NAFLD Non Alcoholic Fatty Liver Disease
  • NAFLD activity level refers to NAFLD progression and is defined by an increase in the steatosis score, as defined herein. NAFLD activity level also refers to of NAFLD progression towardsNASH or Fibrosis and NASH severity According to the present invention, the terms “NAFL” or “Non Alcoholic Fatty Liver” refers to a condition in which fat is deposited in the liver (hepatic steatosis), without signs of inflammation and fibrosis, in the absence of excessive alcohol consumption.
  • the term “steatosis” refers to the process describing the abnormal retention of lipids or fat accumulation within the liver.
  • the term “NASH” or “Non-Alcoholic SteatoHepatitis” refers to a NAFLD condition characterized by the concomitant presence of liver steatosis, hepatocyte ballooning and liver inflammation at histological examination, (i.e. NAS > 3, with at least 1 point in steatosis, at least 1 point in lobular inflammation and at least 1 point in the hepatocyte ballooning scores) in the absence of excessive alcohol consumption and after excluding other liver diseases like viral hepatitis (HCV, HBV).
  • HCV viral hepatitis
  • NASH activity level also refers to NASH progression towards irreversible NASH and/or fibrosis and NASH severity.
  • the term "Active-NASH” refers to a NASH characterized by a NAS>4, with at least 1 point in steatosis score, at least 1 point in the lobular inflammation score and at least 1 point in the hepatocyte ballooning score.
  • hepatocellular ballooning is usually defined, at the light microscopic level, based on hematoxylin and eosin (H&E) staining, as cellular enlargement 1.5-2 times the normal hepatocyte diameter, with rarefied cytoplasm. It refers more generally to the process of hepatocyte cell death.
  • H&E hematoxylin and eosin
  • lobular inflammation refers to the presence of lobular inflammatory foci (grouped inflammatory cells) at microscopic examination of a hematoxylin and eosin (H&E) stained slice of a liver biopsy.
  • H&E hematoxylin and eosin
  • NAFLD-Activity score refers to the sum of steatosis, hepatocellular ballooning, lobular inflammation scores, as follows:
  • Ballooning degeneration score 0: none; 1 : few; 2: many cells/prominent ballooning.
  • the "Activity index” refers to the sum of hepatocellular ballooning and lobular inflammation scores.
  • the term “fibrosis” or “liver fibrosis” refers to the presence of fibrous connective tissue at microscopic examination of a stained (H&E, trichrome or picrosirius red staining) slice of a liver biopsy.
  • the term “fibrosis stage” denotes the localization and extent of fibrosis at histological exam, as follows:
  • the fibrosis stage may be referred to as follows in the context of the present invention:
  • F 4: severe fibrosis (i.e. cirrhosis).
  • To-Be-Treated subject or "TBT subject” is a subject whose disease activity score (e.g. NAS or Activity Index) and/or liver fibrosis stage make the subject eligible to a treatment for NAFLD, NAFL, NASH and/or liver fibrosis (such as for NAFLD, NASH and/or liver fibrosis).
  • a "Not-To-be-treated subject” or “NTBT subject” is a subject whose disease activity score (e.g. NAS or Activity Index) and/or liver fibrosis stage is not high enough to deserve treatment for NAFLD, NAFL, NASH and/or liver fibrosis (such as for NAFLD, NASH and/or liver fibrosis).
  • a TBT subject is also referred to as “receiver” or “potential receiver” for a NAFLD, NASH and/or liver fibrosis treatment (such as for a NAFLD, NASH and/or liver fibrosis treatment).
  • preferential TBT subjects are:
  • the definition encompasses various NASH activity scores and fibrosis stages defining different variants of the invention.
  • Preferential variants of the invention are detailed as follows.
  • TBT2 First TBT variant
  • a TBT2 subject is defined as a subject presenting the following liver biopsy-derived grades:
  • - fibrosis stage ⁇ 2 (such as a fibrosis stage equal to 2, 3 or 4, in particular 2 or 3).
  • a NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • a TBT1 subject is defined as a subject presenting the following liver biopsy-derived grades:
  • - fibrosis stage ⁇ 1 (such as a fibrosis stage equal to 1 , 2, 3 or 4).
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • a TBT7 subject is defined as a subject presenting the following liver biopsy-derived grades:
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • the miR-132 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-132-3p and hsa-miR-132-5p, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0000426 (SEQ ID NO:1 ) and MIMAT0004594 (SEQ ID NO:2) respectively.
  • the miR-132 microRNA implemented in the present invention is a miR-132 stem-loop form, also named HGNC:MIR132, whose sequence is available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0000449 (SEQ ID NO:3).
  • the miR-125 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-125b-5p, hsa-miR-125b-1 -3p, hsa-miR-125b- 2-3p; hsa-miR-125a-5p and hsa-miR-125a-3p whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0000423 (SEQ ID NO:4), MIMAT0004592 (SEQ ID NO:5), MIMAT0004603 (SEQ ID NO:6), MIMAT0000443 (SEQ ID NO:7), MIMAT0004602 (SEQ ID NO:8) respectively.
  • the miR-125 microRNA implemented in the present invention is a miR-125 stem-loop form, such as a miR-125 microRNA selected from the group consisting of hsa-miR-125a, also named HGNC:MIR125A, hsa-miR-125b1 , also named HGNC:MIR125B1 , and hsa-miR-125b2, also named HGNC:MIR125B2, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MI0000469 (SEQ ID NO:9), MI0000446 (SEQ ID NO:10), and MI0000470 (SEQ ID NO:1 1 ) respectively.
  • the miR-505 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-505-5p and hsa-miR-505-3p whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0004776 (SEQ ID NO:12), and MIMAT0002876 (SEQ ID NO:13) respectively.
  • the miR-505 microRNA implemented in the present invention is a miR-505 stem-loop form, such as a miR-505 microRNA selected from the group consisting of hsa-miR-505, also named HGNC:MIR505, whose sequence is available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0003190 (SEQ ID NO:14).
  • the miR-365 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-365a-5p, hsa-miR-365a-3p, hsa-miR-365b-5p, and hsa-miR-365b-3p whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0009199 (SEQ ID NO:15), MIMAT0000710 (SEQ ID NO:16); MIMAT0022833 (SEQ ID NO:17), and MIMAT0022834 (SEQ ID NO:18) respectively.
  • the miR-365 microRNA implemented in the present invention is a miR-365 stem-loop form, such as a miR-365 microRNA selected from the group consisting of hsa-miR-365-1 , also named HGNC:MIR365-1 and hsa-miR-365-2, also named HGNC:MIR365-2, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0000767 (SEQ ID NO:19), and MI0000769 (SEQ ID NO:20) respectively.
  • the miR-22 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-22-5p, and hsa-miR-22-3p, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0004495 (SEQ ID NO:21 ), and MIMAT0000077 (SEQ ID NO:22) respectively.
  • the miR-22 microRNA implemented in the present invention is a miR-22 stem-loop form, such as a miR-22 microRNA, also named hsa-miR-22 or HGNC:MIR22, whose sequence is available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0000078 (SEQ ID NO:23).
  • the miR-378 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-378a-5p, hsa-miR-378a-3p, hsa-miR-378b, hsa-miR-378c, hsa-miR-378d, hsa-miR-378e, hsa-miR-378f, hsa-miR-378g, hsa-miR-378h, hsa-miR-378i, and hsa-miR-378j, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0000731 (SEQ ID NO:24), MIMAT0000732 (SEQ ID NO:25), MIMAT0014999 (SEQ ID NO:26), MIMAT0016847 (SEQ ID NO:27), MIMAT0018926 (SEQ
  • the miR-378 microRNA implemented in the present invention is a miR-378 stem-loop form, such as a miR-378 microRNA selected from the group consisting of hsa-miR-378a, also named HGNC:MIR378, hsa-miR-378b, also named HGNC:MIR378B, hsa-miR-378c, also named HGNC:MIR378C, hsa-miR-378d-1 , also named HGNC:MIR378D1 , hsa-miR-378d-2, also named HGNC:MIR378D2, hsa-miR-378e, also named HGNC:MIR378E, hsa-miR-378f, also named HGNC:MIR378F, hsa-miR-378g, also named HGNC:MIR378G, hsa-miR-378h, also named
  • the miR-320 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-320a-3p, hsa-miR-320b-3p, hsa-miR-320c-3p, hsa-miR-320d-3p, and hsa-miR-320e-3p, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0000510 (SEQ ID NO:46), MIMAT0005792 (SEQ ID NO:47), MIMAT0005793 (SEQ ID NO:48), MIMAT0006764 (SEQ ID NO:49), and Ml MATO015072 (SEQ ID NO:50) respectively.
  • the miR-320 microRNA implemented in the present invention is a miR-320 stem-loop form, such as a miR-320 microRNA, also named hsa-miR-320a or HGNC:MIR320A, hsa-miR-320b-1 or HGNC:MIR320B1 , hsa-miR320c-1 or HGNC:MIR320C1 , hsa-miR-320b-2 or HGNC:MIR320B2, hsa-miR-320d-1 or HGNC:MIR320D1 , hsa-miR-320c-2 or HGNC:MIR320C2, hsa-miR-320d-2 or HGNC:MIR320D2; and hsa-miR-320e or HGNC:MIR320E, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers
  • the miR-885 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-885-5p, and hsa-miR-885-3p, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0004947 (SEQ ID NO:59), and MIMAT0004948 (SEQ ID NO:60) respectively.
  • the miR-885 microRNA implemented in the present invention is a miR-885 stem-loop form, such as a miR-885 microRNA, also named hsa-mir-885 or HGNC:MIR885, whose sequence is available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0005560 (SEQ ID NO:61 ).
  • the miR-483 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-483-5p, and hsa-miR-483-3p, whose sequences are available from the miRBase database (http://mirbase.org) under the miRBase Accession numbers MIMAT0004761 (SEQ ID NO:62, and MIMAT0002173 (SEQ ID NO:63) respectively.
  • the miR-483 microRNA implemented in the present invention is a miR-483 stem-loop form, such as a miR-483 microRNA, also named hsa-mir-483 or HGNC:MIR-483, whose sequence is available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0002467 (SEQ ID NO:64).
  • the miR-30, miR-422, miR-100, or miR-4324, microRNA implemented in the present invention is selected from the group consisting of hsa-miR-30a- 5p, hsa-miR-30a-3p, hsa-miR-30b-5p, hsa-miR-30b-3p, hsa-miR-30c-5p, hsa-miR-30c-1 -3p, hsa-miR-30c-2-3p, hsa-miR-30d-5p, hsa-miR-30d-3p, hsa-miR-30e-5p, hsa-miR-30e-3p, hsa-miR-422a-5p, or hsa-miR-4324-3p whose sequences are available from the miRBase database (http://mirbase.org) respectively under the miRBase Accession numbers MIMAT00000
  • the miR-30 microRNA implemented in the present invention is a miR-30 stem-loop form, such as a miR-30a microRNA, also named hsa-mir-30a or HGNC:MIR30A, whose sequence is available from the miRBase database (http://mirbase.org) under the miRBase Accession number MI0000088 (SEQ ID NO:80), a miR-30b microRNA, also named hsa-mir-30b or HGNC:MIR30B, whose sequence is available from the miRBase database (http://mirbase.org) respectively under the miRBase Accession number MI0000441 (SEQ ID NO:81 ), a miR-30c1 microRNA, also named hsa-mir- 30c1 or HGNC:MIR30C1 , whose sequence is available from the miRBase database (http://mirbase.org) respectively under the miRBase Accession number MI0000736 (SEQ ID NO:82), a miR-30
  • miR-193 is also detected.
  • the miR-452 microRNA implemented in the present invention is selected from the group consisting of hsa-miR-452-5p, hsa-miR-452-3p, whose sequences are, respectively:
  • the miR-452 microRNA implemented in the present invention is a miR-452 stem-loop form, also named HGNC:MIR452, whose sequence is GCUAAGCACUUACAACUGUUUGCAGAGGAAACUGAGACUUUGUAACUAUGUCUCAGU CUCAUCUGCAAAGAAGUAAGUGCUUUGC
  • the term "body fluid sample” denotes any body fluid sample obtained from a subject such as blood and blood-derived fluids (such as plasma and serum), lymphatic fluid, cerebrospinal fluid, synovial fluid, urine, saliva, mucous, phlegm and sputum .
  • the body fluid is selected from blood and blood-derived fluids (such as plasma and serum), saliva, cerebrospinal fluid and urine.
  • the body fluid sample is a blood or blood-derived fluid (such as plasma and serum), saliva, cerebrospinal fluid or urine.
  • the body fluid is blood, plasma or serum.
  • a body fluid sample may be collected by any suitable means.
  • Suitable body fluids may be acellular fluids. Such acellular body fluids are generally produced by processing a cell-containing body fluid by, for example, centrifugation or filtration, to remove the cells. Typically, an acellular body fluid contains no intact cells however, some may contain cell fragments or cellular debris.
  • the body fluid sample may be used immediately or may be stored for later use. Any suitable storage method known in the art may be used to store the body fluid sample: for example, the sample may be frozen at about - 20° C to about -80° C. miRNA isolation and quantification
  • Total RNA including miRNA can be purified from a sample by various methods of extraction which include either: phenokchloroform extraction followed by alcohol precipitation (TRIzol), phenokchloroform followed by solid-phase extraction (column-based; e.g. miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II) magnetic particles, or direct lysis methods.
  • phenokchloroform extraction followed by alcohol precipitation (TRIzol) phenokchloroform followed by solid-phase extraction (column-based; e.g. miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II) magnetic particles, or direct lysis methods.
  • miRNA were extracted with miRVana Paris extraction kit for subsequent RTqPCR analysis or captured with specific probes for further HTG Edge Sequence analysis
  • miRNAs are detected in clinical samples using any technique available to those skilled in the art, such as sequencing-based, amplification-based, or hybridization-based methods.
  • Common approaches to miRNA clinical testing include small RNA sequencing (Hafner et al, 2012; Vigneault et al, 2012), HTG Edge Whole Transcriptome assay, a next-generation sequencing-based miRNA profiling platform (Lizarraga et al, 2016; Satake et al, 2018), quantitative miRNA real-time reverse-transcription PCR (qRT-PCR) (Chen et al, 2005), miRNA microarray (Castoldi et al, 2007), multiplexed miRNA detection with color-coded probe pairs (NanoString n Counter expression system) (Geiss et al, 2008), droplet digital PCR (ddPCR) after reverse transcription (Miotto et al, 2014), and miRNA in situ hybridization (Nelson et al, 2006).
  • small RNA sequencing
  • the level of a microRNA may be determined by conventional methodologies well known in the art, such as immunoassays (e.g. ELISA), or molecular biology assays (quantitative RT-PCR or Next-Generation-Sequencing) or biochemical assays (colorimetric assays or others).
  • immunoassays e.g. ELISA
  • molecular biology assays quantitative RT-PCR or Next-Generation-Sequencing
  • biochemical assays colorimetric assays or others.
  • miRNA are detected by HTG Edge whole transcriptome assays or HTG Edge sequencing, and RT-qPCR.
  • any of the above described methods may further comprise normalizing the level of the assayed microRNA in the body fluid sample from the subject and in the reference to the level or a microRNA whose level does not vary in NAFLD, NASH and/or liver fibrosis subjects relative to healthy patients.
  • a spike-in or exogenous synthetic micro-RNA of known sequence and quantity such as C. elegans miR-39, may be added to the sample before RNA extraction.
  • the spike-in or exogenous synthetic micro-RNA may be a miRNA that is not expressed in human samples, such as Caenorhabditis elegans cel-miR-38 or Arabidopsis thaliana ath- miR-159a.
  • These synthetic micro-RNA may be added after addition of the lysis buffer in blood derived samples before RNA extraction and provide a process control for technical normalization. The efficiency of RNA extraction, complementary DNA synthesis and PCR amplification can be therefore monitored using these exogenous synthetic micro-RNAs
  • a micro-RNA normalizer or small non coding RNA controls for the normalization of qPCR data representing endogenous controls that are affected by the same sources of variability as the target genes, during all the steps of the experimental pipeline, may be used to normalize the level of the target miRNA.
  • a standard protocol for measuring a microRNA by quantitative RT-PCR is provided. Briefly, the measures are carried out from total RNA extracted from a body fluid sample such as blood, plasma or serum sample, in particular a cell-free, citrate-derived platelet-free plasma sample.
  • An appropriate internal control such as a micro-RNA of known sequence and quantity, e.g. C. elegans miR-39
  • Cq values are determined using quantitative RT-PCR.
  • the Taqman miRNA RT-qPCR assay Taqman MicroRNA Reverse transcription Kit, TaqMan MicroRNA Assay 20X, and TaqMan Universal Master Mix II (Applied Biosystems) may be used according to the manufacturer's instructions.
  • Reverse transcription may be performed using readily available PCR systems, such as the GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems), with appropriate cycling parameters such as 16°C for 30 minutes followed by 42°C for 30 minutes and 85°C for 5 minutes before holding at 4°C.
  • the reverse transcription may be implemented in the multiplexed format.
  • Quantitative PCR is then conducted using a quantitative PCR system such as the CFX96TM Real-Time System (C1000 TouchTM Thermal Cycler, BioRad).
  • a quantitative PCR system such as the CFX96TM Real-Time System (C1000 TouchTM Thermal Cycler, BioRad).
  • quantitative PCR is conducted using a CFX96-Real-Time PCR Detection System - C1000 - In Vitro Diagnostic (IVD) certified, Bio-Rad. Cycling conditions may be the following: 95°C for 10 minutes followed by 95°C for 15 sec and 60°C for 60 sec for a total of 50 cycles and then 30°C for 30 sec.
  • Cq determination mode may be, for example, the Regression mode in the quantitative PCR system.
  • the Cq value determined according to the method of the invention is the Cq value which is obtainable using the above specific parameters and material.
  • Cq values of samples may be excluded from the analysis if values are above the maximum Cq of the standard curve of each miRNA.
  • the standard curve may be used to assess the PCR reaction efficiency. Serial dilutions may be performed over eight points starting from the most concentrated cDNA sample, to ensure the standard curve covers all potential template concentrations that may be encountered during the study.
  • the standard curve may be constructed by plotting the log of the starting quantity of the template against the Cq values obtained.
  • RNA extracted from serum samples may be used for reverse transcription concurrently with RNA extracted from serum samples.
  • the product may then be serially diluted and PCR may be performed on all samples (standards and serum- derived RNA). Standard curve may be performed in simplicate, duplicate or triplicate and used to convert Cq data in copies ⁇ L of fluid.
  • delta Ct Cycle threshold
  • delta Cq Cycle quantification
  • the level of the microRNA may be determined by RT-qPCR using stem-loop reverse transcription (RT) reaction combined with TaqMan qPCR, or with a poly(A)-tailed RT combined with SYBR Green detection and Lock Nucleic Acid (LNA) primers.
  • RT stem-loop reverse transcription
  • LNA Lock Nucleic Acid
  • a preferred embodiment relates to the determination of the level of at least one microRNA selected from the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100 and miR-4324, in a blood, serum or plasma sample.
  • a preferable variant of this aspect relates to the determination of the level of at least one microRNA selected in the group consisting of hsa-miR-132-3p, miR-125b-5p, hsa-miR-125b-2-3p, hsa- miR-505-3p, hsa-miR-365-3p, hsa-miR-22-3p, hsa-miR-378a-3p, hsa-miR-320e-3p, hsa- miR-885-5p, hsa-miR-483-5p, hsa-miR-30a-5p, hsa-miR-422a, hsa-miR-100-5p and hsamiR- 4324-3p.
  • the present invention relates to a method for the diagnosis or detection of a NAFLD in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for the diagnosis or detection of a potential NAFLD in a subject, comprising determining the level of said miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample of said subject.
  • NAFLD or potential NAFLD is detected based on increased level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324 in the body fluid sample from the subject, relative to a reference level measured in a sample from a subject with no hepatic steatosis.
  • the diagnosis or detection of NAFLD or potential NAFLD is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample relative to levels generally measured in healthy subjects with no hepatic steatosis.
  • the method further comprise a step of confirming that the subject suffers from NAFLD.
  • Such confirmation may be implemented according to any method known by those skilled in the art, such as by conducting a liver biopsy or by ultrasound or imaging techniques (such as ultrasonography, controlled attenuation parameter measurement by transient elastography (Fibroscan), Magnetic Resonance Imaging (MRI), MRI-estimated proton density fat fraction (MRI-DPFF), and the Magnetic resonance spectroscopy density fat fraction (MRS-DPFF)).
  • ultrasound or imaging techniques such as ultrasonography, controlled attenuation parameter measurement by transient elastography (Fibroscan), Magnetic Resonance Imaging (MRI), MRI-estimated proton density fat fraction (MRI-DPFF), and the Magnetic resonance spectroscopy density fat fraction (MRS-DPFF)
  • MRI-DPFF Magnetic Resonance Imaging
  • MRS-DPFF Magnetic resonance spectroscopy density fat fraction
  • FLI fatty liver index
  • GTT gamma glutaryl transferase
  • HIS hepatic steatosis index
  • AST serum aspartate aminotransferase
  • ALT alanine aminotransferase ratio
  • BMI gender and presence of diabetes mellitus
  • NAFLD liver fat score metabolic syndrome, type 2 diabetes, fasting serum insulin and AST, AST:ALT ratio
  • the steatotest (alpha 2 Macroglobulin (A2M), Haptoglobin, apolipoprotein A1 , Total Bilirubin, GGT, fasting blood gluose and adjustment for age, sex, weight and height), and
  • ALT cholesterol, triglycerides, glycated hemoglobin A1 c (HbA1 c) and leukocyte count
  • HbA1 c glycated hemoglobin A1 c
  • leukocyte count comorbidity data
  • genetic and genomic markers may assess NAFLD risk and severity (Single Nucleotide Polymorphisms (SNPs):rs738409 (SNP in PNPLA3), cell-free non coding RNAs, miR-122, composite panel of serum derived omics data).
  • SNPs Single Nucleotide Polymorphisms
  • rs738409 Single Nucleotide Polymorphisms
  • miR-122 single Nucleotide Polymorphisms
  • composite panel of serum derived omics data composite panel of serum derived omics data
  • the present invention relates to a method for the diagnosis or detection of a NAFL in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for the diagnosis or detection of a potential NAFL in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324, in a body fluid sample of said subject.
  • NAFL or potential NAFL is detected based on increased level of miR- miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid sample from the subject, relative to a reference level measured in a sample from a subject with no hepatic steatosis.
  • the diagnosis or detection of NAFL or potential NAFL is based on the detection of an increased level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample relative to levels generally measured in healthy subjects with no hepatic steatosis.
  • the method further comprise a step of confirming that the subject suffers from NAFL.
  • Such confirmation may be implemented according to any method known by those skilled in the art, such as by conducting a liver biopsy or by ultrasound or imaging techniques (such as ultrasonography, controlled attenuation parameter measurement by transient elastography (Fibroscan), Magnetic Resonance Imaging (MRI), MRI-estimated proton density fat fraction (MRI-DPFF), and the Magnetic resonance spectroscopy density fat fraction (MRS-DPFF)).
  • ultrasonography controlled attenuation parameter measurement by transient elastography (Fibroscan), Magnetic Resonance Imaging (MRI), MRI-estimated proton density fat fraction (MRI-DPFF), and the Magnetic resonance spectroscopy density fat fraction (MRS-DPFF)
  • MRI-DPFF Magnetic Resonance Imaging
  • MRS-DPFF Magnetic resonance spectroscopy density fat fraction
  • FLI fatty liver index
  • GTT gamma glutaryl transferase
  • HIS hepatic steatosis index
  • AST serum aspartate aminotransferase
  • ALT alanine aminotransferase
  • BMI gender and presence of diabetes mellitus
  • NAFLD liver fat score metabolic syndrome, type 2 diabetes, fasting serum insulin and AST, AST:ALT ratio
  • the steatotest (alpha 2 Macroglobulin (A2M), Haptoglobin, apolipoprotein A1 , Total Bilirubin, GGT, fasting blood gluose and adjustment for age, sex, weight and height), and
  • ALT cholesterol, triglycerides, glycated hemoglobin A1 c (HbA1 c) and leukocyte count
  • HbA1 c glycated hemoglobin A1 c
  • leukocyte count comorbidity data
  • genetic and genomic markers may assess NAFLD risk and severity (Single Nucleotide Polymorphisms (SNPs):rs738409 (SNP in PNPLA3), cell-free non coding RNAs, miR-122, composite panel of serum derived omics data).
  • SNPs Single Nucleotide Polymorphisms
  • rs738409 Single Nucleotide Polymorphisms
  • miR-122 single Nucleotide Polymorphisms
  • composite panel of serum derived omics data composite panel of serum derived omics data
  • the present invention also relates to a method for the diagnosis or detection of a NASH in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for the diagnosis or detection of a potential NASH in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the diagnosis or detection of NASH or of potential NASH is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the body fluid from the subject, relative to a reference level of the same microRNA measured in a healthy subject, in a subject with NAS ⁇ 3 or in a subject with at least one component of NAS scored at 0.
  • the diagnosis or detection of NASH or potential NASH is based on the detection of an increased expression level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in blood, serum or plasma relative to reference levels measured in non-NASH subjects including healthy subject, subjects with NAS ⁇ 3 or subjects with at least one component of NAS scored at 0.
  • the method further comprises a step of confirming that the subject suffers from NASH.
  • Such confirmation may be implemented according to any method known by those skilled in the art, such as by conducting a liver biopsy or by imaging biomarkers measured by imaging techniques such as MRI based techniques, gadoxetic acid used with MRI, super paramagnetic iron oxide MRI, Intracellular ATP level using 32 P-MRS and MRE.
  • imaging techniques such as MRI based techniques, gadoxetic acid used with MRI, super paramagnetic iron oxide MRI, Intracellular ATP level using 32 P-MRS and MRE.
  • imaging techniques such as MRI based techniques, gadoxetic acid used with MRI, super paramagnetic iron oxide MRI, Intracellular ATP level using 32 P-MRS and MRE.
  • imaging techniques such as MRI based techniques, gadoxetic acid used with MRI, super paramagnetic iron oxide MRI, Intracellular ATP level using 32 P-MRS and MRE.
  • indices and scores may assess potential NASH biomarkers, including, without limitation:
  • CK18 fragment CK18 fragment, total cytokeratin, serum levels of apoptosis-mediating surface antigen FAS
  • CRP C-reactive protein
  • TNF TNF
  • IL-8 CXC chemokine ligand 10
  • CXCL10 CXC chemokine ligand 10
  • adipocytokines and hormones adiponectin, leptin, resistin, visfatin, retinol binding protein (RBP)4, fatty acid binding protein (FABP)4, fibroblast growth factor (FGF21 )
  • RBP retinol binding protein
  • FGF21 fibroblast growth factor
  • the present invention also relates to a method for the diagnosis or detection of Active-NASH in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR- 22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for the diagnosis or detection of a potential Active-NASH in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the diagnosis or detection of Active-NASH or of potential Active- NASH is based on the detection of an increased level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324 in the body fluid from the subject, relative to a reference level of the same microRNA measured in a healthy subject, in a subject with NAS ⁇ 4 or in a subject with at least one component of NAS scored at 0.
  • the diagnosis or detection of Active-NASH or potential Active-NASH is based on the detection of an elevated expression level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR- 320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in blood, serum or plasma samples of a subject compared to reference levels measured in healthy subjects, subjects with NAS ⁇ 4 or subjects with at least one component of NAS scored at 0.
  • the method further comprises a step of confirming that the subject suffers from Active-NASH.
  • Such confirmation may be implemented according to any method known by those skilled in the art, such as by conducting a liver biopsy or by imaging techniques such as MRI based techniques, super paramagnetic iron oxide MRI, multiparemetric MRI, MRS and MRE.
  • imaging techniques such as MRI based techniques, super paramagnetic iron oxide MRI, multiparemetric MRI, MRS and MRE.
  • several indices and scores may assess potential NASH biomarkers, including, without limitation:
  • CK18 fragment CK18 fragment, total cytokeratin, serum levels of apoptosis-mediating surface antigen FAS
  • CRP C-reactive protein
  • TNF TNF
  • IL-8 CXC chemokine ligand 10
  • CXCL10 CXC chemokine ligand 10
  • adipocytokines and hormones adiponectin, leptin, resistin, visfatin, retinol binding protein (RBP)4, fatty acid binding protein (FABP)4, fibroblast growth factor (FGF21 )
  • RBP retinol binding protein
  • FGF21 fibroblast growth factor
  • NASH test NASH diagnostic panel
  • NAFLD risk progression towards NASH or Fibrosis
  • severity markers like genetic and genomic markers like SNPs (rs738409 in PNPLA3), cell-free non coding RNAs (miR-122, miR-1290, miR-192 and miR- 7b), composite panel of serum derived omics data like rs738409 and proteomic data including ACY1 , SHBG, CTSZ, MET, GNS, LGALS3BP, CHL1 and SERPINC1 , SNPs at multiple loci (PNPLA3, SOD2, KLF6 and LPIN1 ), miR-122, composite panel including miR- 122, miR-192, miR-21 , ALT, CK18 Asp396, cell free DNA like circulating methylated PPARG.
  • the present invention also relates to a method for characterizing the occurrence or grade of liver lobular inflammation in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR- 100, or miR-4324 in a body fluid sample of said subject.
  • the present invention also relates to a method for characterizing the occurrence or grade of hepatocyte ballooning in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR- 100, or miR-4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for characterizing the occurrence or grade of liver steatosis in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for the diagnosis or detection of liver fibrosis in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR- 22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the present invention also relates to a method for the diagnosis or detection of a potential liver fibrosis in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422, miR-100, or miR-4324 in a body fluid sample of said subject.
  • the fibrosis is at minimum a significant fibrosis (i.e. F > 2).
  • the diagnosis or detection of liver fibrosis or of potential liver fibrosis is based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR- 22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324 in the body fluid from the subject, relative to a reference level of the same microRNA measured in a subject with no or minimal fibrosis, in particular with minimal fibrosis.
  • the fibrosis is at minimum a moderate liver fibrosis or cirrhosis (i.e. F > 3).
  • the diagnosis or detection of liver fibrosis or of potential liver fibrosis is based on the detection of an increased level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324 in the body fluid from the subject, relative to a reference level of the same microRNA measured in a subject with no fibrosis, with minimal fibrosis, or with severe fibrosis, in particular with severe fibrosis.
  • the method further comprises a step of confirming that the subject suffers from liver fibrosis, or confirming the stage of liver fibrosis.
  • confirmation may be implemented according to any method known by those skilled in the art, such as by conducting a liver biopsy or by imaging biomarkers, including, without limitation:
  • liver fibrosis and cirrhosis were tested for liver fibrosis and cirrhosis.
  • FIB-4 index (FIB-4) which comprises age, AST, ALT, and platelet count
  • NAFLD fibrosis score (age, BMI, impaired fasting glucose and/or diabetes, AST, ALT, platelet count, and albumin),
  • the BARD core (AST, ALT, BMI, and diabetes).
  • liver fibrosis markers and panel may assess liver fibrosis: - Specific fibrosis markers: Hyaluronic acid, N-terminal pro-peptide of collagen type III (PIIINP), neo epitope specific competitive enzyme linked immunosorbent assay for PIIINP (Pro-C3), Tissue Inhibitor Metalloproteinase 1 (TIMP-1 ), Laminin.
  • PIIINP N-terminal pro-peptide of collagen type III
  • Pro-C3 neo epitope specific competitive enzyme linked immunosorbent assay for PIIINP
  • Tissue Inhibitor Metalloproteinase 1 Tissue Inhibitor Metalloproteinase 1
  • EEF Enhanced Liver Fibrosis
  • PIIINP PIIINP
  • Hyaluronic acid PIIINP
  • TIMP-1 TIMP-1
  • Fibrotest gamma glutamyl transferase (GGT), total bilirubin, alpha 2 macroglobulin (A2M), apolipoprotein A1 and haptoglobin
  • FibroMeter NAFLD body weight, prothrombin index, ALT, AST, ferritin and fasting glucose.
  • the present invention also relates to a method for the determination of liver fibrosis stage in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the method for determining the stage of liver fibrosis further comprises a step of confirming the stage of liver fibrosis in the subject.
  • Such confirmation may be implemented according to any method known by those skilled in the art, such as by conducting a liver biopsy or by other means like imaging biomarkers listed above for the diagnosis of fibrosis.
  • the present invention also relates to diagnosis and detection of significant or advanced liver fibrosis due to other fibrotic liver diseases such as: viral hepatitis (HBV, HCV,..), Alcoholic steatohepatitis, Biliary diseases (Primary biliary cholangitis, Primary sclerosing cholangitis, Autoimmune hepatitis, Wilson's disease, Alphal antitrypsine deficiency).
  • viral hepatitis HBV, HCV,..
  • Alcoholic steatohepatitis Alcoholic steatohepatitis
  • Biliary diseases Primary biliary cholangitis, Primary sclerosing cholangitis, Autoimmune hepatitis, Wilson's disease, Alphal antitrypsine deficiency.
  • the present invention also relates to a method for classifying a subject as a potential receiver or non-receiver treatment for NAFLD, NASH and/or liver fibrosis, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the method is for classifying the subject as a potential receiver or non-receiver treatment for NAFLD.
  • the method is for classifying the subject as a potential receiver or non-receiver treatment for NASH.
  • the method is for classifying the subject as a potential receiver or non- receiver treatment for liver fibrosis.
  • the present invention also relates to a method for classifying a subject as a potential receiver or non-receiver treatment for NAFL, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the method is for classifying the subject as a potential receiver or non-receiver treatment for NAFL.
  • the method is for classifying the subject as a potential receiver or non-receiver treatment for NAFL.
  • the method is for classifying the subject as a potential receiver or non-receiver treatment for liver fibrosis.
  • the present invention more particularly relates to a method for classifying a subject as a potential receiver (TBT) or non-receiver (NTBT) of a treatment for NASH and/or fibrosis, comprising determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • TBT potential receiver
  • NBT non-receiver
  • a subject is classified as a TBT2 subject if the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 in the body fluid sample from said subject is higher than the level of the same microRNA in a reference sample of a NTBT2 subject.
  • a subject is classified as a TBT1 subject if the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 in the body fluid sample from said subject is higher than the level of the same microRNA in a reference sample of a NTBT1 subject.
  • a subject is classified as a TBT7 subject if the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 in the body fluid sample from said subject is higher than the level of the same microRNA in a reference sample of a NTBT7 subject.
  • the method of the invention is for classifying a subject as a TBT2 subject.
  • variants of the invention relates to a method for classifying patients as being potential receiver (TBT) or non-receiver (NTBT) of a treatment for NASH and/or fibrosis, based on the detection of an elevated expression level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in blood, serum or plasma compared to reference levels of the same microRNA measured in NTBT patients.
  • Such a classification may also be the basis for determining whether a subject should undergo further liver investigations, such as state-of-the-art liver investigations, before taking decision to treat, such as ultrasound, elastography, imaging techniques including MRI, or liver biopsy.
  • the definition of TBT or receiver vs NTBT or non-receiver patient may vary depending on the drug efficacy to safety of drug with varying disease activity values (NAS or activity Index) and varying fibrosis stage value as provided above.
  • the present invention also relates to a method for the determination of a NAFLD or NASH activity in a subject, comprising determining the level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324, in a body fluid sample of said subject.
  • the invention also relates to a method for the prognostic of the risk of NAFLD or NASH activity evolution in a subject, comprising determining the level of miR-132, miR-125, miR- 505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample of said subject.
  • the method is for the prognostic of the risk of NAFLD or NASH activity evolution in absence of a treatment.
  • the present invention also relates to a method for the determination of liver fibrosis stage in a subject, comprising determining the level of of miR-132, miR-125, miR-505, miR-365, miR- 22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the invention also relates to a method for the prognostic of the risk of fibrosis evolution to cirrhosis and liver clinical outcomes in a subject, comprising determining the level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the method is for the prognostic of the risk fibrosis evolution to cirrhosis and liver clinical outcomes in the absence of a treatment.
  • the invention also relates to a method for monitoring the evolution (i.e. progression or regression) of NAFLD or NASH activity in a subject, comprising determining the level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the invention also relates to a method for monitoring the evolution (i.e.
  • liver fibrosis in a subject, comprising determining the level of miR-132, miR- 125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the invention also relates to a method for predicting the response of a patient to a specific treatment of NAFLD, NASH and/or liver fibrosis, comprising determining the level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample of said subject.
  • the invention also relates to a method for predicting the response of a patient to a specific treatment of NAFL, comprising determining the level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324, in a body fluid sample of said subject.
  • the invention relates to a method for the diagnosis and detection of NAFLD in a subject, based on the detection of an increased level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324, in a body fluid sample relative to levels generally measured in healthy subjects with no hepatic steatosis.
  • the invention relates to a method for the diagnosis and detection of NAFL in a subject, based on the detection of an increased level of miR-132, miR-125, miR-505, miR-365, miR- 22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324, in a body fluid sample relative to levels generally measured in healthy subjects with no hepatic steatosis, no hepatic ballooning and no lobular inflammation.
  • the invention relates to a method for the diagnosis and detection of NASH in a subject, based on the detection of an increased expression level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324, in blood, serum or plasma relative to reference levels measured in non-NASH subjects including healthy subject, subjects with NAS ⁇ 3 or subjects with at least one component of NAS scored at 0.
  • the invention relates to a method for the diagnosis and detection of Active-NASH in a subject, based on the detection of an elevated expression level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100, or miR-4324, in blood, serum or plasma samples of a subject compared to reference levels measured in healthy subjects, subjects with NAS ⁇ 4 or subjects with at least one component of NAS scored at 0.
  • the invention relates to a method for determination of steatosis stage in a subject, based on the detection of the level of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324, in a body fluid sample of a subject.
  • the invention relates to a method for determination of hepatocellular ballooning grade in a subject, based on the detection of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324, in a body fluid sample of a subject.
  • the invention relates to a method for determination of lobular inflammation grade in a subject, based on the detection of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR- 100, or miR-4324, in a body fluid sample of a subject.
  • cut-off concentrations of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 may be calculated to help the decision-making by the person implementing the methods of the present invention.
  • the expression "cut-off concentration" as used herein refers to a concentration of the microRNA above which a statistical prediction of a symptom or disease is made, and below which a statistical prediction of a lack of a disease or symptom is made. Such cut-off concentrations may be determined as follows for different scenarios.
  • ROC Receiveiver Operating Characteristics
  • a cut-off concentration for classifying a subject as a subject with NASH (or potential NASH) or as a subject without NASH can be determined by:
  • ROC Receiveiver Operating Characteristics
  • a cut-off concentration for classifying a subject as a subject with an Active-NASH (or potential Active-NASH) or as a subject without an Active-NASH subject can be determined by:
  • ROC Receiveiver Operating Characteristics
  • a cut-off concentration for classifying a subject as a subject with significant liver fibrosis (F > 2) (or potential significant liver fibrosis) or as a subject with no or minimal fibrosis can be determined by:
  • miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 concentrations in body fluid samples of reference cohorts of subjects including both subjects with significant to severe liver fibrosis (F > 2) or advanced liver fibrosis (F > 3) and subjects with no or minimal fibrosis (F 0-1 ), ii) applying a dedicated statistical analysis to the reference data set to determine an optimal cut-off concentration.
  • ROC Receiveiver Operating Characteristics
  • a cut-off concentration for classifying a subject as a TBT subject or as a NTBT subject can be determined by:
  • ROC Receiveiver Operating Characteristics
  • miR-132, miR-125, miR-505, miR-365, miR-22, miR- 378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is a circulating diagnostic biomarker for non-invasive grading of histological lesions (steatosis, lobular inflammation, hepatocyte ballooning), assessment of NAFLD activity level, NASH activity level and assessment of liver fibrosis severity in a subject.
  • a method to prognostic the risk of NAFLD or NASH activity evolution in a subject in the absence of a treatment based on the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324, in a body fluid sample of a subject.
  • Another variant of the invention relates to a method to prognostic the risk of fibrosis evolution to cirrhosis and liver outcomes of a NAFLD or NASH patient based on the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 measured in a body fluid sample of a subject.
  • the present invention is also dedicated to prognostic the risk of fibrosis evolution in patients suffering from other fibrotic liver diseases such as: viral hepatitis (HBV, HCV,..), Alcoholic steatohepatitis, Biliary diseases (Primary biliary cholangitis, Primary Sclerosing cholangitis, Autoimmune hepatitis, Wilson's disease, Alphal antitrypsine deficiency).
  • viral hepatitis HBV, HCV,..
  • Alcoholic steatohepatitis Alcoholic steatohepatitis
  • Biliary diseases Primary biliary cholangitis, Primary Sclerosing cholangitis, Autoimmune hepatitis, Wilson's disease, Alphal antitrypsine deficiency.
  • the inventors have also shown that there is a correlation between changes in circulating levels of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100, or miR-4324 and evolution of histological scores, notably evolution of the Activity Index, NAS and fibrosis stage.
  • the method of the invention can be used for assessing the anti- NAFLD, anti-NASH and/or anti-fibrotic activity of a drug in interventional trials assuming changes in serum level miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 as surrogates of histological evolutions.
  • another variant of the invention relates to a method for monitoring the evolution (i.e. progression or regression) of NAFLD or NASH activity based on the evolution of the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422, miR-100, or miR-4324 in body fluid samples collected two or more times apart from the same subject.
  • Another variant of the invention relates to a method for monitoring the evolution (i.e.
  • liver fibrosis stage based on the evolution of the level of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in body fluid samples collected two or more times apart from a same subject.
  • the present invention is also dedicated to the determination of fibrosis stage evolution in other fibrotic liver diseases such as: viral hepatitis (HBV, HCV,..), Alcoholic steatohepatitis, Biliary diseases (Primary biliary cholangitis, Primary Sclerosing cholangitis, Autoimmune hepatitis, Wilson's disease, Alphal antitrypsine deficiency).
  • viral hepatitis HBV, HCV,..
  • Alcoholic steatohepatitis Alcoholic steatohepatitis
  • Biliary diseases Primary biliary cholangitis, Primary Sclerosing cholangitis, Autoimmune hepatitis, Wilson's disease, Alphal antitrypsine deficiency.
  • Another variant of the invention relates to a method for predicting the response of a subject (prediction of changes in NAFLD activity, NASH activity and liver fibrosis stage) to a specific treatment (responder subject) based on the detection of a differential expression level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422a, miR-100, or miR-4324 in a body fluid sample of the subject compared to reference levels measured in non-responder subjects.
  • liver steatosis characterize the occurrence of liver steatosis in a subject. Furthermore, according to the present invention, methods are provided to:
  • liver fibrosis and/or a more advanced liver fibrosis stage are diagnose the subject to have liver fibrosis and/or a more advanced liver fibrosis stage
  • liver steatosis and/or more advanced liver steatosis score.
  • classify a subject as a receiver or non-receiver of a treatment for liver fibrosis classify a subject as a receiver or non-receiver of a treatment for hepatocellular ballooning
  • a subject classify a subject as a receiver or non-receiver of a treatment for lobular inflammation, or
  • a subject classify a subject as a receiver or non-receiver of a treatment for liver steatosis.
  • the methods for determining whether a subject has NAFLD or NASH, or Active-NASH or liver fibrosis (such as significant liver fibrosis), or lobular inflammation, or hepatocyte ballooning or for determining if a subject is a drug receiver (TBT) or a potential responder to a specific drug comprise collecting a sample of a body fluid from a subject suspected of having the assessed condition, and detecting the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR- 100, or miR-4324, wherein a level that is higher than a reference level of the respective miRNA, indicates the presence of the assessed condition, or the diagnosis of the subject as having NAFLD or NASH, or Active-NASH or liver fibrosis (such as significant liver fibrosis), or lobular inflammation, or hepatocyte ballooning or the subject
  • TBT
  • the subject is a subject at risk of having NALFD, NASH, Active- NASH or liver fibrosis or a subject at risk of developing NAFLD, NASH, Active-NASH or liver fibrosis in the future, such as a subject having obesity, diabetes, suffering from the metabolic syndrome, and/or having elevated liver enzymes and/or having other signs of liver dysfunctions.
  • the subject may also be a subject with previously identified NAFLD, NASH or Active-NASH or liver fibrosis, the method of the invention thereby allowing determining the disease activity and fibrosis stage and estimating risks of evolution of the disease towards cirrhosis, cirrhotic complications, hepatocarcinoma, liver transplantation, a cardiovascular or liver-related deaths.
  • the subject is suffering from NASH, the method of the invention thereby allowing determining the efficacy of a drug for the treatment of the NASH disease, classifying the subject as responder/non-responder to a treatment for NASH, or monitoring the evolution of the NASH state of the subject.
  • the level of at least two microRNA selected in the group consisting of miR-132, miR-125, miR- 505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least three microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least four microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least five microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least six microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 is determined.
  • the level of at least seven microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least eight microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least nine microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least ten microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least eleven microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of at least twelve microRNA selected in the group consisting of miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 is determined.
  • the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100 and miR-4324 is determined.
  • the measure of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 level can be introduced in mathematical models (algorithms) for combination with other variables such as sex, age, body mass index, weight, medical status, arterial pressure or other body fluid markers such as blood, serum or
  • the methods of the present invention comprise the determination of the level of other biomarkers in addition to miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • biomarkers are selected from the group consisting of: alpha 2 macroglobulin (A2M), glycated haemoglobin (HbAl c), fasting glucose level or fructosamine level, N-terminal pro-peptide of collagen type III (PIIINP) and YKL-40.
  • A2M alpha 2 macroglobulin
  • HbAl c glycated haemoglobin
  • PIIINP N-terminal pro-peptide of collagen type III
  • YKL-40 YKL-40.
  • such biomarker is YKL-40.
  • such biomarkers are NAFLD, NASH or liver fibrosis markers, such as the degree of steatosis, necroinflammation and fibrosis, estimated by Magnetic Resonance Imagery (MRI), Magnetic Resonance Elastography (MRE), Magnetic Resonance Spectroscopy (MRS), Controlled attenuation parameter (CAP) and liver stiffness measurement by Transient Elastography (TE), Ultrasonography (USG), FibroScan, Point Shear Wave Elastography (pSWE), 2D Shear Wave Elastography (2D-SWE), Single Nucleotide Polymorphisms (SNP), cell free DNA, cell free non coding RNA, and gene polymorphisms (such as PNPLA3 and TM6SF2).
  • MRI Magnetic Resonance Imagery
  • MRE Magnetic Resonance Elastography
  • MRS Magnetic Resonance Spectroscopy
  • CAP Controlled attenuation parameter
  • TE Transient Elastography
  • USG Ultrasonography
  • such biomarkers are NAFLD markers like fatty liver index related markers, Hepatic steatosis index related markers, NAFLD liver fat score related markers, SteatoTest parameters, NAFLD ridge score parameters, circulating triglycerides, Body Mass Index (BMI); imaging biomarkers like the degree of beam scattering by the tissue (USG), the degree of ultrasound attenuation by hepatic fat (CAP), the proton density fat fraction (MRI- PDFF), the liver triglyceride content, signal fat fraction (MRS).
  • NAFLD markers like the degree of beam scattering by the tissue (USG), the degree of ultrasound attenuation by hepatic fat (CAP), the proton density fat fraction (MRI- PDFF), the liver triglyceride content, signal fat fraction (MRS).
  • such biomarkers are NASH biochemical blood markers like apoptosis markers (CK18 fragment, total cytokeratin, serum levels of apoptosis-mediating surface antigen FAS), inflammatory markers (C-reactive protein (CRP), TNF, IL-8, CXC chemokine ligand 10 (CXCL10)), lipid oxidation products ( 1 1 -hydroxyeicosatetraenoic acid (HETE), 9-hydroxydecadienoic acid (HODE), 13-HODE, 13-oxo-octadecadienoic acid (ODE), LA-13-HODE (oxNASH score), 1 1 ,12-dihydroxy-eicosatrienoic acid (diHETrE)), adipocytokines and hormones (adiponectin, leptin, resistin, visfatin, retinol binding protein (RBP)4, fatty acid binding protein (FABP)4, fibroblast growth factor (FGF21), NASH biochemical blood
  • such biomarkers are liver fibrosis markers: imaging biomarkers like mechanically induced impulse, quantitative measurement of shear wave speed (FibroScan-transient elastography, pSWE-ARFI, 2D-3D-SWE), ultrasound induced focused radiation force impulse at death (pSWE-ARFI), use of modified phase-contrast method to image the propagation of the shear wave in liver parenchyma (MRE); biochemical bloodmarkers like the AST:ALT ratio, the AST:platelet ratio index (APRI), the FIB4 index parameters, the NAFLD fibrosis score parameters, the BARD score parameters, specific fibrosis markers like HA, PIIINP, Pro-C3, TIMP-1 , Laminin, ELF related panels, fibrotest parameters, fibroMeter NAFLD parameters.
  • imaging biomarkers like mechanically induced impulse, quantitative measurement of shear wave speed (FibroScan-transient elastography, pSWE-ARFI, 2D-3D-SWE), ultrasound induced
  • markers are NAFLD risk and severity markers like genetic and genomic markers like SNPs (rs738409 in PNPLA3), cell-free non coding RNAs (miR-122, miR-1290, miR-192 and miR-7b), composite panel of serum derived omics data like rs738409 and proteomic data including ACY1 , SHBG, CTSZ, MET, GNS, LGALS3BP, CHL1 and SERPINC1 , SNPs at multiple loci (PNPLA3, SOD2, KLF6 and LPIN1 ), miR-122, composite panel including miR-122, miR-192, miR-21 , ALT, CK18 Asp396, cell free DNA like circulating methylated PPARG.
  • SNPs rs738409 in PNPLA3
  • cell-free non coding RNAs miR-122, miR-1290, miR-192 and miR-7b
  • composite panel of serum derived omics data like rs738409 and prote
  • the other biomarkers are other circulating microRNAs in addition to miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100, or miR-4324.
  • additional microRNAs that may be useful in the practice of the present invention include: miR-34a, miR- 122, or miR-200.
  • circulating microRNAs whose level could be further determined include miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p
  • miR-452 such as hsa-miR-452, in particular hsa
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of at least one microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hs
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR- 452-5p) is determined, in addition to the level of at least two microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hs
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa- miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of at least three microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p) is determined, in addition to the level of at least four microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of at least five microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hs
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR- 452-5p) is determined, in addition to the level of at least six microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hs
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa- miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of at least seven microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p) is determined, in addition to the level of at least eight microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR- 885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of at least nine microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hs
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR- 452-5p) is determined, in addition to the level of at least ten microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, h
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa- miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of at least eleven microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p) is determined, in addition to the level of at least twelve microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-
  • the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a- 5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR- 452 (such as hsa-miR-452, in particular hsa-miR-452-5p) is determined, in addition to the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100 and miR-4324.
  • the methods may comprise the steps of:
  • the diagnosis, detection, monitoring, evaluation of the risk or evaluation of the efficacy of a treatment for NAFLD, NASH or liver fibrosis is conducted by determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in a body fluid sample of the subject, and submitting the subject to physical, non-invasive, techniques such as ultrasound, elastography or imaging techniques such as MRI.
  • physical, non-invasive, techniques such as ultrasound, elastography or imaging techniques such as MRI.
  • the methods of the present invention may be combined to the method disclosed in WO2017046181 owned by the same Applicant.
  • a decision may be taken to give life style recommendations to a subject (such as a food regimen or providing physical activity recommendations), to medically take care of a subject (e.g. by setting regular visits to a physician or regular examinations, for example for regularly monitoring markers of liver damage), or to administer at least one NAFLD, NASH or liver fibrosis therapy to a subject.
  • a decision may be taken to give life style recommendations to a subject or to administer at least one NAFLD, NASH or liver fibrosis therapy.
  • Such a classification of a subject as a receiver or TBT patient is based on an elevated level on miR- 132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, and miR-4324 compared to reference miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, and miR-4324, levels measured in non-receiver patients (NTBT), as provided above.
  • NTBT non-receiver patients
  • the invention thus further relates to an anti-NAFLD, anti-NASH or anti-fibrotic compound for use in a method for treating NAFLD, NASH or liver fibrosis in a subject in need thereof, wherein the subject has been identified thanks to a method according to the invention.
  • the invention thus further relates to an anti-NAFL compound for use in a method for treating NAFLD, NASH or liver fibrosis in a subject in need thereof, wherein the subject has been identified thanks to a method according to the invention.
  • the invention relates to an anti-NAFLD compound for use in a method for treating NAFLD in a subject in need thereof, wherein the subject has been classified as a receiver of said treatment thanks to a method according to the invention.
  • the invention relates to an anti-NASH compound for use in a method for treating NASH in a subject in need thereof, wherein the subject has been classified as a receiver of said treatment thanks to a method according to the invention.
  • the invention relates to an anti-fibrotic compound for use in a method for treating liver fibrosis in a subject in need thereof, wherein the subject has been classified as a receiver of said treatment thanks to a method according to the invention.
  • X1 represents a halogen, a R1 , or G1 -R1 group
  • X2 represents a G2-R2 group
  • G1 and G2 identical or different, represent an atom of oxygen or sulfur
  • R1 represents a hydrogen atom, an unsubstituted alkyl group, an aryl group or an alkyl group that is substituted by one or more halogen atoms, an alkoxy or an alkylthio group, cycloalkyl groups, cycloalkylthio groups or heterocyclic groups;
  • R2 represents an alkyl group substituted by at least a -COOR3 group, wherein R3 represents a hydrogen atom, or an alkyl group that is substituted or not by one or more halogen atoms, cycloalkyl groups, or heterocyclic groups.
  • Adenosine A3 receptor agonists like 2-(1 -Hexynyl)-N-methyladenosine, Piclidenoson CF- 101 (IB-MECA), Namodenoson CF-102, 2-CI-IB-MECA, CP-532,903, Inosine, LUF-6000, and MRS-3558.
  • Apararenone MT 3995
  • Amiloride Spironolactone
  • Eplerenone Eplerenone
  • Canrenone and potassium canrenoate
  • progesterone progesterone
  • drospirenone gestodene
  • benidipine benidipine
  • - AMP activated protein kinase stimulators like PXL-770, MB-1 1055 Debio-0930B metformin, CNX-012, O-304, mangiferin calcium salt, eltrombopag, carotuximab, and Imeglimin.
  • Amylin receptor agonist and Calcitonin receptor agonists include, but are not limited to, KBP-042 and KBP-089.
  • Angiopoietin-related protein-3 inhibitors like ARO-ANG3, IONIS-ANGGPTL3-LRx or AKCEA-ANGPTL3LRx, evinacumab, and ALN-ANG.
  • Antisense oligonucleotide targeting transforming growth factor beta 2 include, but are not limited to ASPH-0047, IMC-TR1 and ISTH-0047.
  • Bile acids includelike obeticholic acid (OCA) and UDCA, norursodeoxycholic acid, and ursodiol.
  • Bioactive lipids like 5-hydroxyeicosapentaenoic acid (15-HEPE, DS-102), unsaturated fatty acids such as 25 arachidonic acid, icosapentethyl ester, eicosapentaneoic acid, and docosahexaenoic acid.
  • - Cannabinoid CB1 receptor antagonists like GRC-10801 , MRI-1569, MRI-1867, DBPR-21 1 , AM-6527, AM-6545, NESS-1 1 -SM, CXB-029, GCC-2680, TM-38837, Org-50189, PF- 514273, BMS-812204, ZYO-1 , AZD-2207, AZD-1 175, otenabant, ibipinabant,surinabant ; rimonabant, drinabant, SLV-326, V-24343, and O-2093.
  • Dual cannabinoid CB1 receptor/iNOS inhibitor Caspase inhibitors like emricasan, belnacasan, nivocasan, IDN-7314, F-573, VX-166, YJP- 60107, MX-1 122, IDN-6734, TLC-144, SB-234470, IDN-1965, VX-799, SDZ-220-976, and L- 709049.
  • - Cathepsin inhibitors like VBY-376, VBY-825, VBY-036, VBY-129, VBY-285, Org-219517, LY3000328, G-7236, and BF/PC-18.
  • CCR antagonists like cenicriviroc (CCR2/5 antagonist), PG-092, RAP-310, INCB-10820, RAP-103, PF-04634817, and CCX-872.
  • DGAT Diacylglycerol-O-acyltransferase inhibitors like IONIS-DGAT2Rx formely ISIS- DGAT2RX, ISIS 703802, LY-3202328, BH-03004, KR-69530, OT-13540, AZD-7687, PF-
  • IONIS-DGAT2Rx formely ISIS- DGAT2RX, ISIS 703802, LY-3202328, BH-03004, KR-69530, OT-13540, AZD-7687, PF-
  • DPP4 Dipeptidyl peptidase IV
  • evogliptin vidagliptin, fotagliptin, alogliptin, saxagliptin, tilogliptin, anagliptin, sitagliptin, retagliptin, melogliptin, gosogliptin,trelagliptin, teneligliptin, dutogliptin, linagliptin, gemigliptin, yogliptin, betagliptin, imigliptin, omarigliptin, vidagliptin, and denagliptin.
  • DPP4 Dipeptidyl peptidase IV
  • NADPH oxidase NOX (NADPH oxidase) inhibitors, like Dual NOX (NADPH oxidase) 1 &4 inhibitors; GKT-831 (2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1 H-pyrazolo[4,3-c]pyridine- 3,6(2H,5H)-dione),formely GKT137831 , and GKT-901.
  • Fatty Acid Synthase (FAS) inhibitors like TVB-2640; TVB-3664; TVB-3166, TVB-3150, TVB-3199, TVB-3693BZL-101 , 2-octadecynoic acid, MDX-2, Fasnall, MT-061 , G28UCM, MG-28, HS-160, GSK-2194069, KD-023, and cilostazol.
  • FOS Fatty Acid Synthase
  • the FAS inhibitor is a compound selected in the following list of compounds:
  • the FAS inhibitor is selected from:
  • the FAS inhibitor is TVB-2640.
  • omega-3 fatty acids like omega-3 fatty acids, Omacor or MF4637, fish oils, poly unsatured fatty acids (efamax, optiEPA).
  • FABAC fatty acid bile acid conjugates
  • FXR Farnesoid X receptor
  • FGF-19 Fibroblast Growth Factor 19 receptor ligand or functional engineered variant of FGF-19
  • FGF-19 Fibroblast Growth Factor 19
  • FGF-21 Fibroblast Growth Factor 21 agonists like PEG-FGF21 formely BMS-986036, YH- 25348, BMS-986171 , YH-25723, LY-3025876, and NNC-0194-0499.
  • Galectin 3 inhibitors like GR-MD-02, TD-139, ANG-4021 , Galectin-3C, LJPC-201 , TFD-100, GR-MD-03, GR-MD-04, GM-MD-01 , GM-CT-01 , GM-CT-02, Gal-100, and Gal-200.
  • GLP-1 Glucagon-like peptide-1 analogs like semaglutide, liraglutide, exenatide, albiglutide, dulaglutide, lixisenatide, loxenatide, efpeglenatide, taspoglutide, MKC-253, DLP-
  • GLP-1 Glucagon-like peptide-1 receptor agonists like LY-3305677, and Oxyntomodulin long acting.
  • GPCR G-protein coupled receptor
  • GPR84 antagonist G-protein coupled receptor 84 antagonist
  • FFAR1 agonist Free fatty acid receptor 1 agonist
  • Hedgehog cell-signalling pathway inhibitors like Vismodegib, TAK-441 , IPI-926, Saridegib, Sonidegib/Erismodegib, BMS-833923/XL139, PF-04449913, Taladegib/LY2940680, ETS- 2400, SHR-1539, and CUR61414.
  • Ileal sodium bile acid cotransporter inhibitors like A-4250, GSK-2330672, volixibat, CJ- 14199, and elobixibat.
  • - Insulin sensitizer and MCH receptor-1 antagonist like MSDC-0602k, MSDC-0602, CSTI-100 and AMRI.
  • Integrin inhibitors integrin inhibitors of Pliant Therapeutic, integrin inhibitors of Indalo Therapeutics, integrin inhibitors of St Louis University, ProAgio, and GSK-3008348.
  • Ketohexokinase inhibitors like JNJ-28165722; JNJ-42065426; JNJ-42152981 ; JNJ- 42740815; JNJ-42740828, and PF-06835919.
  • LT Leukotriene
  • PDE Phosphodiesterase
  • LO Lipoxygenase
  • Lysyl oxidase homolog 2 inhibitors like Rappaport, InterMune, Pharmaxis, AB-0023, Simtuzumab, PXS-5382A, and PXS-5338.
  • Macrolides likesolithromycin, azithromycin, and erythromycin .
  • Macrophage mannose receptor modulators like AB-0023, MT-1001 , [18F]FB18mHSA, Xemys, technetium Tc 99m tilmanocept, and CDX-1307.
  • - Methyl CpG binding protein 2 modulator and transglutaminase inhibitors include, but are not limited to, cysteamine, EC Cysteamine, enteric-coated cysteamine bitartrate, cysteamine bitartrate (enteric-coated), Bennu, cysteamine bitartrate (enteric-coated), Raptor, cysteamine bitartrate, DR Cysteamine, delayed release enteric coated cysteamine bitartrate, mercaptamine, mercaptamine (enteric-coated), Bennu, mercaptamine (enteric-coated), Raptor, RP-103, RP-104, PROCYSBI, and mercaptamine (enteric-coated).
  • - miRNA antagonists like RG-125 formely AZD4076, RGLS-5040, RG-101 , MGN-5804, and MRG-201 .
  • MMP9 - Metalloproteinase-9
  • Mitochondrial carrier family inhibitor and Mitochondrial phosphate carrier protein inhibitor include, but are not limited to TRO-19622, Trophos, olesoxime, RG-6083, or RO-7090919.
  • - Myeloperoxidase inhibitors include, but are not limited to PF-06667272
  • GS-6624 mAb against LOXL2, ustekinumab an anti-TNF antibody, and inebilizumab.
  • Monoclonal antibodies like anti-IL20 mAbs, anti-TGF3 antibodies, anti-CD3 antibodies, anti-LOXL2 antibodies and anti-TNF antibodies.
  • GPR109 Nicotinic Acid Receptor
  • NTZ N- nitazoxanide
  • TZ active metabolite tizoxanide
  • RM-5061 prodrugs of TZ
  • NSAIDs non-steroid anti-inflammatory drugs
  • F-351 salicylates (aspirin), acetaminophen, propionic acid derivatives (ibuprofen, naproxen), acetic acid derivatives (indomethacin, diclofenac), enolic acid derivatives (piroxicam, phenylbutazone), anthranilic acid derivatives (meclofenalmic acid, flufenamic acid), selective COX-2 inhibitors (celecoxib, parecoxib), and sulfonanilides (nimesulide).
  • salicylates aspirin
  • acetaminophen propionic acid derivatives
  • acetic acid derivatives indomethacin, diclofenac
  • enolic acid derivatives piroxicam, phenylbutazone
  • anthranilic acid derivatives meclofenalmic acid, flufenamic acid
  • selective COX-2 inhibitors celecoxib,
  • Phenylalanine hydroxylase stimulators like Pegvaliase, sapropterin, AAV-PAH, CDX-61 14, sepiapterin, RMN-168, ALTU-236, ETX-101 , HepaStem, rolipram, and alprostadil.
  • PAR-2 antagonists like PZ-235, and NP-003.
  • - PPAR alpha agonists like fenofibrate, ciprofibrate, pemafibrate, gemfibrozil, clofibrate, binifibrate, clinofibrate, clofibric acid, nicofibrate, pirifibrate, plafibride, ronifibrate, theofibrate, tocofibrate, and SR10171 ;
  • - PPAR gamma agonists like Pioglitazone, deuterated pioglitazone, Rosiglitazone, efatutazone, ATx08-001 , OMS-405 , CHS-131 , THR-0921 , SER-150-DN, KDT-501 , GED- 0507-34-Levo, CLC-3001 , and ALL-4.
  • GW501516 Endurabol or ( ⁇ 4-[( ⁇ 4-methyl-2-[4- (trifluoromethyl)phenyl]-1 ,3-thiazol-5-yl ⁇ methyl)sulfanyl]-2-methylphenoxy ⁇ acetic acid)
  • MBX8025 Seladelpar or ⁇ 2-methyl-4-[5-methyl-2-(4-trifluoromethyl- phenyl)-2H-[l,2,3]triazol- 4-ylmethylsylfanyl]-phenoxy ⁇ -acetic acid
  • GW0742 [4-[[[2-[3-fluoro-4-
  • glitazars like Saroglitazar, Aleglitazar, Muraglitazar, Tesaglitazar, DSP-8658.
  • CLA conjugated linoleic acid
  • PPAR alpha/gamma/delta agonists or PPAR pan agonists like IVA337 (Lanifibranor), TTA (tetradecylthioacetic acid), Bavachinin, GW4148, GW9135, Bezafibrate, Lobeglitazone, and CS038.
  • Rho-associated protein kinase 2 (ROCK2) inhibitors like KD-025, TRX-101 , BA-1049, LYC- 53976, INS-1 17548, and RKI-1447.
  • ASK1 Signal-regulating kinase 1
  • SGLT Sodium-glucose transport 1 inhibitors like LX-4212/LX-421 1/sotagliflozin, SAR - 439954, LIK-066 (Licoglifozin), LX-2761 , GSK-161235, LP-925219, KGA-2727, SAR-7226, SAR-474832, SY-008, and AVX-3030.
  • SGLT Sodium-glucose transport 2 inhibitors like remogliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, ipraghflozin, tianagliflozin, canagliflozin, tofogliflozin, janagliflozin, bexagliflozin, luseogliflozin, sergliflozin, HEC-44616, AST-1935, and PLD-101 .
  • Thyroid receptor ⁇ Thyroid receptor ⁇ (THR ⁇ ) agonists likeVK-2809, MGL-3196, MGL-3745, SKL-14763, sobetirome, BCT-304, ZYT-1 , MB-0781 1 , and eprotirome.
  • TLR-2 Toll Like Receptor 2 and 4 (TLR-2) antagonists like CI-201 also known as VB-201 .
  • TLR-4 Toll Like Receptor 4 antagonists like naltrexone, JKB-121 , M-62812, resatorvid, dendrophilin, CS-4771 , AyuV-1 , AyuV-25, NI-0101 , EDA-HPVE7, and eritoran.
  • RTK Tyrosine kinase receptor
  • VAP-1 Vascular adhesion protein-1
  • AOC3 Amine Oxidase Copper containing 2
  • VDR Vitamin D receptor
  • alfacalcidol 1 ,25-dihydroxyvitamin D3, Vitamin D2, Vitamin D3, calcitriol, Vitamin D4, Vitamin D5, dihydrotachysterol, calcipotriol, tacalcitol 1 ,24- dihydroxyvitamin D3, and paricalcitol.
  • Vitamin E and isoforms Vitamin E combined with vitamin C and atorvastatin.
  • anti-NASH agents include KB-GE-001 and NGM-386 and NGM-395 and NC-10 and TCM-606F. Further anti-NASH agents include icosabutate, NC-101 , NAIA-101 colesevelam, and PRC-4016. Other anti-fibrotic agents include HEC-585, INV-240, RNAi therapeutic (Silence Therapeutics) and SAMiRNA program (Bioneer Corp).
  • illustrative antifibrotic agents include pirfenidone or receptor tyrosine kinase inhibitors (RTKIs) such as Nintedanib, Sorafenib and other RTKIs, or angiotensin II (AT1 ) receptor blockers, or CTGF inhibitor, or any antifibrotic compound susceptible to interfere with the TGF3 and BMP-activated pathways including activators of the latent TGF3 complex such as MMP2, MMP9, THBS1 or cell-surface integrins, TGF3 receptors type I (TGFBRI) or type II (TGFBRII) and their ligands such as TGF3, Activin, inhibin, Nodal, anti-Mijllerian hormone, GDFs or BMPs, auxiliary co-receptors (also known as type III receptors), or components of the SMAD-dependent canonical pathway including regulatory or inhibitory SMAD proteins, or members of the SMAD-independent or non-canonical pathways including various branches
  • the treatment of NASH or liver fibrosis comprises administering a compound of formula (I) selected in the group consisting of 1 -[4-methylthiophenyl]-3-[3,5- dimethyl-4-carboxydimethylmethyloxy phenyl]prop-2-en-1 -one, 1 -[4-methylthiophenyl]-3- [3,5-dimethyl-4-isopropyloxy carbonyldimethylmethyloxyphenyl]prop-2-en-1 -one, 1 -[4- methylthiophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl] prop-2- en-1 -one, 1 -[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyl dimethylmethyloxyphenyl]prop-2-en-1 -one, 1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-
  • the compound of formula (I) is 1 -[4- methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxy phenyl]prop-2-en-1 -one or a pharmaceutically acceptable salt thereof.
  • the invention relates to a combination product comprising at least an anti- NAFLD, and/or an anti-NASH, and/or an anti-Fibrotic agent for use in a method for treating NAFLD, NASH, active NASH, and/or Liver fibrosis in a subject in need thereof, wherein the subject has been classified as a receiver of said treatment thanks to a method according to the invention.
  • the invention relates to the treatment of NAFLD, NASH, Active NASH, and/or Liver fibrosis with a combination product comprising at least one agent selected from the group of anti-NAFLD, anti-NASH and/or anti-fibrotic compounds, or pharmaceutically acceptable salts thereof.
  • the invention relates to the treatment of NAFLD, NASH, Active NASH, and/or Liver fibrosis with Elafibranor.
  • NASH or liver fibrosis comprises administering NTZ, TZ, vitamin E or pioglitazone, obeticholic acid, elafibranor, selonsertib, saroglitazar and/or cenicrivoc.
  • the treatment of NASH or liver fibrosis comprises administering NTZ or TZ, in particular NTZ.
  • a combination treatment is conducted.
  • the treatment of NAFLD, NAFL, NASH, Active NASH, or Liver fibrosis comprises administering Elafibranor combined with one or more other anti-NAFLD, anti- NAFL, anti-NASH or anti-liver fibrosis compound.
  • the treatment of NAFLD, NAFL, NASH, Active NASH, or Liver fibrosis comprises administering Elafibranor combined with at least one compound selected in the group consisting of NTZ, TZ, vitamin E or pioglitazone, obeticholic acid, elafibranor, selonsertib, saroglitazar and cenicrivoc.
  • the treatment of NAFLD, NAFL, NASH, Active NASH, or Liver fibrosis comprises administering Elafibranor combined with NTZ.
  • the results obtained by the inventors also support pathophysiological roles of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324 in the development and evolution of NAFLD, NASH and/or liver fibrosis.
  • the results obtained by the inventors also support pathophysiological roles of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324 in the development and evolution of NAFL.
  • the methods of the invention thus can be used to identify specific subpopulations of subjects with NAFLD, NASH and/or liver fibrosis based on circulating levels of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR- 100, or miR-4324.
  • miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR- 4324 dependent disease which would make these patients responsive to specific drugs acting directly (miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 mimics, deregulator of miRNA like circular RNA (CircRNA) or anti-miR-132, anti-miR-125, anti-miR-505, anti-miR-365, anti-miR- 22, anti-miR-378, anti-miR-320, anti-miR-885, anti-miR-483, anti-miR-30, anti-m
  • the methods of the invention thus can be used to identify specific subpopulations of subjects with NAFL based on circulating levels of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324.
  • the invention relates to a miR-132, miR- 125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 inhibitor compound for use in the treatment of NAFLD, NASH or liver fibrosis in a subject in need thereof.
  • miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 inhibitor compound refers to any compound, such as a nucleic acid compound, able to prevent the action of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 and particularly of miR-132-5p, miR-132-3p, hsa-miR-125a-5p, hsa-miR-125a-3p, hsa-miR-125b-5p, hsa-miR-125b-1 -3p and hsa-miR-125b-2
  • the miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324 inhibitor compound of the present invention is a compound that inhibits or reduces the activity of the respective miRNA, for example by binding to miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 or that inhibits the respective miRNA expression.
  • inhibiting miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 expression means that the production of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422a, miR-100, or miR-4324 in the liver or hepatocytes after treatment with said inhibiting compound is less than the amount produced prior to treatment.
  • miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR- 422a, miR-100, or miR-4324 expression has been inhibited in liver or hepatocytes, using for example techniques for determining miRNA transcript level.
  • Suitable miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR- 483, miR-30, miR-422a, miR-100, or miR-4324 inhibitor compounds include double or single-stranded RNA (such as short- or small-interfering RNA or "siRNA"), antagomirs, antisense nucleic acids, circular RNA, artificial miRNA sponges and enzymatic RNA molecules such as ribozymes. Each of these compounds can be targeted to a given miRNA and destroy or induce the destruction of the target miRNA.
  • siRNA short- or small-interfering RNA or "siRNA”
  • RNA interference of the miRNA can be inhibited by inducing RNA interference of the miRNA with an isolated double-stranded RNA ("dsRNA") molecule which has at least 90%, for example 95%, 98%, 99% or 100%, sequence homology with at least a portion, or preferably with the entirety, of the miRNA.
  • the dsRNA molecule is a siRNA.
  • siRNAs useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length.
  • the siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base- paired").
  • the present invention relates to a kit comprising means for determining the level of:
  • the present invention also relates to a kit comprising means for determining the level of:
  • the kit comprises means for determining the level of at least one microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least two microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least three microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least four microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least five microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least six microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a-3
  • the kit comprises means for determining the level of at least seven microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least eight microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least nine microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least ten microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193
  • the kit comprises means for determining the level of at least eleven microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of at least twelve microRNA selected in the group consisting of miR-132, miR-125, miR-505, miR- 365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR- 4324, and optionally means for determining the level of miR-193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b-5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa-miR-452, in particular hsa- miR-452-5p).
  • miR-193 such as hsa-miR-193, in particular hsa-miR-193a
  • the kit comprises means for determining the level of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR- 30, miR-422, miR-100, and miR-4324, and optionally means for determining the level of miR- 193 (such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b- 5p and hsa-miR-193b-3p, more particularly hsa-miR-193b-3p) and/or miR-452 (such as hsa- miR-452, in particular hsa-miR-452-5p).
  • miR- 193 such as hsa-miR-193, in particular hsa-miR-193a-3p, hsa-mi
  • the kit of the invention is useful for implementing the methods described above. It may further optionally include instructions for implementing said methods.
  • the kit may comprise reagents and buffers appropriate for conducting measures of the levels of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR- 100, or miR-4324 and any other circulating marker of liver damage as provided above.
  • the kit may comprise antibodies specific for a protein to be quantified, and/or primers useful
  • the kit may comprise primers and/or probes for quantifying micro-RNA levels, as well-known in the art.
  • the kit may comprise reagents and buffers appropriate for conducting measures of the levels of miR-132, miR-125, miR-505, miR-365, miR-22, miR-378, miR-320, miR-885, miR-483, miR-30, miR-422, miR-100, or miR-4324 and any other marker of NAFLD and/or NASH.
  • the kit of the invention is useful for implementing the methods described above and may further optionally include instructions for implementing said methods.
  • the kit may comprise reagents and buffers appropriate for conducting measures of the levels of markers identified above.
  • the kit may comprise antibodies specific for a protein to be quantified, and/or primers useful for quantifying micro-RNA levels, as well-known in the art.
  • the kit comprises means for determining the level of miR-132-3p, miR-125b-5p, miR-125b-2-3p, miR-505-3p, miR-365-3p, miR-22-3p, miR-378a-3p, miR- 320e-3p, miR-885-5p, miR-483-5p, miR-30a-5p, miR-100-5p, miR-422a-5p, and/or miR- 4324-3p.
  • a fibrosing NASH was induced by feeding rats a CDAA diet for 78 days. Supplementation of the CDAA diet with 1 % cholesterol resulted in the development of a more severe NASH pathology with a significant rate of hepatocytes ballooning and high penetration of advanced fibrosis differentiated this model. Animals were sacrificed at the given time, organs and plasma collected.
  • Blood collected in citrate containing tubes was processed by separating cell-free plasma from blood cells within 15 minutes of collection by centrifugation at 1 ,500xg for 15 minutes. The supernatant plasma was transferred to a new tube. Tubes were kept at -70°C. To proceed to RNA extraction, plasma tubes were then centrifuged at 13,000xg for 2 min to pellet and remove the platelets. The supernatant platelet-free plasma (PFP) was transferred to a new tube, frozen in liquid nitrogen and stored at -80°C.
  • PFP platelet-free plasma
  • NAFLD Activity Score was established as the unweighted sum of steatosis grading, lobular inflammation grading and ballooning injury severity. Fibrosis area was quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, USA), as the percentage of collagen-positive stained area relative to the whole image area.
  • nuclease free water was used to adjust the volume of RNA to 8 ⁇ _, to which, 2 ⁇ _ RNA Spike Control Oligos was added.
  • the ATP mixture was diluted in 1 mM Tris and the Poly (A) tailing master mix was prepared according to the Affymetrix protocol. After the addition of 5 ⁇ _ master mix to the 10 ⁇ _ RNA Spike Control Oligos, the mixture was incubated at 37°C for 15 min.
  • RNA was utilized for the ligation step by adding 4 ⁇ _ 5X FlashTaq Biotin HSR ligation mixture followed by 2 ⁇ _ T4 deoxyribonucleic acid (DNA) ligase to each sample, then incubating at room temperature for 30 min. After the reaction was stopped through the addition of 2.5 ⁇ _ HSR stop solution, 23.5 ⁇ _ ligated sample was added.
  • the Enzyme Linked Oligo Sorbent Assay quality control was performed prior to array hybridization according to the manufacturer's procedure. A volume of 21.5 ⁇ _ biotin labeled sample was then used for hybridization on Affymetrix Gene Chip® miRNA 4.0 Arrays.
  • the arrays were then washed and stained with Buffer A, Buffer B, and cocktail 1 and 2 followed by scanning using Affymetrix Command console software (version 1 .3.1 ). After scanning, the raw data files produced at the end of the array scan (CEL files) were extracted. The raw data obtained from Affymetrix were analyzed using Expression Console software (Affymetrix) for quality controls. The regularized t-test implemented in the limma R package was used (FC ⁇ 1 .5; p- value ⁇ 0.05) to detect differential expression between two groups.
  • RNA with preserved miRNAs was extracted from 100 ⁇ of blood derived fluid (human serum and rat PFP) by miRVanaParis extraction kit (AM1556, Ambion, Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Synthetic spiked-in C. elegans miR-39 was added to the samples [3,125 fmoles] (MSY0000010, Qiagen, Venlo, The Netherlands) prior to RNA extraction as internal control of RNA extraction process. The elution was performed in 100 ⁇ of elution buffer.
  • RNA total RNA (5 ⁇ ) was used for reverse transcription (RT) reaction.
  • RT reverse transcription
  • Reverse Transcription was performed using a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems) with cycling conditions of 16°C for 30 minutes followed by 42°C for 30 minutes and 85°C for 5 minutes before holding at 4°C.
  • RNA samples were used as a template for the qPCR assay.
  • Quantitative PCR were performed using a CFX96TM Real-Time System (C1000 TouchTM Thermal Cycler, BioRad, Hercules, CA) with cycling conditions of 95°C for 10 minutes followed 95°C for 15 sec and 60°C for 60 sec for a total of 50 cycles and 30°C for 30 sec.
  • the phase 2 clinical trial GOLDEN-505 (NCT01694849) was a multicentre, randomized, double blind, placebo-controlled study to evaluate the efficacy and safety of Elafibranor (1 -[4- methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxy phenyl]prop-2-en-1 -one) once daily on steatohepatitis in patients with Non-Alcoholic Steatohepatitis (NASH). Liver biopsy was performed to confirm the diagnosis of NASH after appropriate exclusion of liver disease of other etiology. NASH was diagnosed as steatohepatitis evaluated by liver biopsy within 6 months before randomization.
  • Steatohepatitis confirmation was based on central reading of liver biopsies. NASH patients were defined with a NAS ⁇ 3 including steatosis score ⁇ 1 and hepatocyte ballooning ⁇ 1 and lobular inflammation ⁇ 1 . The study was approved by appropriate regulatory bodies all patients had given informed consent for participation.
  • An inclusion liver biopsy was used for examination and scoring of histological lesions. Blood samples were withdrawn at screening and at the end of the 1 -year treatment period for hematology, and clinical biochemistry analysis including a comprehensive list of NAFLD/NASH related parameters. In patients who have signed a dedicated informed consent, additional blood samples were collected for research of new diagnostic biomarkers of NASH. Blood samples used in this biomarker study were drawn from patients of the GOLDEN-DIAG study at inclusion (270 samples) and one year later (223 samples). The inventors had also access to human blood samples from subjects with a liver biopsy and associated clinical and biological data from the UZA Biobank, the OBESE cohort.
  • This cohort which is composed of morbidly obese patients, also comprises NAFLD/non-NASH patients, NASH patients, cirrhotic patients and healthy controls.
  • the serum of 253 patients was processed for the validation of candidate circulating miRNA identified in GOLDEN-DIAG study with next generation sequencing (NGS) technology (HTG EdheSeq) and RT-qPCR respectively.
  • NGS next generation sequencing
  • HGS EdheSeq next generation sequencing
  • RESOLVE-IT is a Multicenter, Randomized, Double-Blind, Placebo-Controlled Phase III Study (NCT02704403) to Evaluate the Efficacy and Safety of Elafibranor in Patients with Nonalcoholic Steatohepatitis (NASH) and fibrosis.
  • NASH Nonalcoholic Steatohepatitis
  • the study was approved by appropriate regulatory bodies all patients had given informed consent for participation. An inclusion liver biopsy was used for examination and scoring of histological lesions. Blood samples were withdrawn at screening. In patients who have signed a dedicated informed consent, additional blood samples were collected for research of new diagnostic biomarkers of NASH.
  • the serum of 370 patients of the RESOLVE-IT study at screening with 263 corresponding liver biopsy was processed for the validation of candidate circulating miRNA identified in GOLDEN-DIAG study with HTG Edge sequence analysis and RTqPCR analysis.
  • the serum of 100 subjects from EFS was processed for the assessment in healthy subjects of candidate circulating miRNA identified in GOLDEN- DIAG study with HTG Edge sequence analysis. Serum samples were used for the HTG Edge sequence analysis.
  • the serum samples of the three cohorts were used for the HTG Edge sequence analysis and RTqPCR analysis.
  • HEMATOLOGY includes hemoglobin, hematocrit, RBC count, leukocytes, differential leukocyte count (neutrophils, lymphocytes, eosinophils, monocytes, basophils -abs. and % values), platelet count and reticulocytes.
  • BIOCHEMISTRY Panel I includes plasma glucose, triglycerides (TG), creatinine, creatinine clearance, gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK), alkaline phosphatase, thyroid stimulating hormone (TSH) and HbA1 c.
  • BIOCHEMISTRY Panel II includes plasma glucose, creatinine, creatinine clearance, total protein, albumin, sodium, potassium, chloride, calcium, uric acid, urea expressed as blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), alkaline phosphatase, creatine phosphokinase (CPK), bilirubin total, bilirubin conjugated, C-reactive protein (hsCRP), AST/ALT Ratio and HbA1 c.
  • BUN blood urea nitrogen
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • GTT gamma-glutamyltransferase
  • CPK creatine phosphokinase
  • hsCRP C-reactive protein
  • AST/ALT Ratio HbA1 c.
  • URINALYSIS includes:
  • Microscopy analysis includes RBC, WBC, casts, crystals, bacteria, epithelial cells and yeasts.
  • SEROLOGY includes HIV ab I/ II, HCV ab, HCV RNA (only tested upon receipt of HCV RNA Visit samples and in case of 'reactive' or 'indeterminate' result for HCV Ab) and HbsAg.
  • LIPID PANEL includes triglycerides (TG), total cholesterol, non HDL-C (calculation), highdensity
  • HDL-C lipoprotein cholesterol
  • LDL-C low density lipoprotein
  • VLDL-C calculated very low density lipoprotein cholesterol (VLDL-C) (calculation), apolipoprotein Al (ApoAI) and apolipoprotein B (ApoB).
  • URINE CHEMISTRY includes alpha-1 -microglobulin, beta-N-acetylglucosaminidase(beta- NAG) and neutrophil-gelatinase associated lipocalin(N-Gal)
  • SAFETY MARKERS includes homocysteine, NT-ProBNP, Troponin T, Cystatin C, and Beta2-microglobulin.
  • GLYCEMIC AND OTHER LIPIDIC PARAMETERS includes leptin, insulin, homeostatic model assesment (HOMA-IR), serum glucose (for calculation of HOMA-IR), fructosamine, Cpeptide and free fatty acids (FFA).
  • INFLAMMATORY MARKERS includes haptoglobin, fibrinogen, tumor necrosis factor alpha (TNF-a), interleukine 6 (IL-6) and plasminogen activator inhibitor 1 (PAI-1 ) Ag (citrate).
  • LIVER MARKERS includes cytokeratin-18 (CK18)(M65 & M30), adinopectin, ferritin, alpha2 macroglobulin, FGF19 & FGF21 , hyaluronic acid (Advia centaur, reagentiaprocured by
  • Blood collected in citrate containing tubes 2.7mL was processed by separating cell-free plasma from blood cells within 15 minutes of collection by centrifugation at 1 ,500xg for 15 minutes. The supernatant plasma was transferred to a new tube. Tubes were kept at -70°C. To proceed to RNA extraction, plasma tubes were then centrifuged at 13,000xg for 2 min to pellet and remove the platelets. The supernatant platelet-free plasma was transferred to a new tube, frozen in liquid nitrogen and stored at -80°C.
  • Blood collected in serum separating tube (SST) 8.5ml_ was processed one hour after 15 sampling by separating cell-free serum from blood cells by centrifugation between 1 ,300xg and 2,000xg for 10 minutes. The serum was then transferred to a new tube. Tubes were kept at -70°C. RNA extraction was performed without additional centrifugation.
  • HTG Edge Sequencing System was used for sequencing the miRNAs contained in human serum samples.
  • Serum levels of 2083 miRNAs were measured using HTG-EdgeSeq-NGS technology.
  • HTG whole transcriptome miRNA (WTA) kit was used.
  • Samples were prepared using 15 ⁇ I of plasma lysis buffer and 15 ⁇ I of plasma sample and 3 ⁇ of Proteinase K are mixed and incubated at 50°C for 60 min with orbital shaking. 25 ⁇ of the mix is transferred to the HTG sample plate and loaded into the HTG processor to perform the nuclease protection assay and prepare the stoichiometric NPP.
  • Barcoding is performed using Hemo KlenTaq enzyme. For each sample, we mix 2.4 ⁇ of Hemo KlenTaq, 0.6 ⁇ of dNTPs (10 nM), 6 ⁇ of OneTaq PCR GC Buffer 5X, 3 ⁇ of Forward and Reverse Primers, 3 ⁇ of sample preparation and 12 ⁇ of H20. In order to remove excess of primer from the library, Agentcour AMPure XP beads were used. Library concentration of for each sample was performed using Kapa Biosystems qPCR Kit. Each sample is pooled in order to generate a pooled library and sequenced on an lllumina NextSeq500. For each sample, at least 250.000 reads are generated. Data reconstruction and analysis were performed using FASTQ files and processed by the HTG Parser software.
  • the objective of the analyses is to discover biomarkers that can be related to the identification of NASH patients to be treated.
  • Patients to be treated are defined differently according to the different parts of the study.
  • TBT2 are defined as:
  • NAS NAFLD Activity Score
  • NAS is defined as the sum of the steatosis score, hepatocyte ballooning score and lobular inflammation grade
  • - fibrosis stage ⁇ 2 (such as a fibrosis equal to 2, 3 or 4, in particular 2 or 3).
  • Quality control checks aim to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing pipelines.
  • HTG whole transcriptome miRNA (WTA) kit was used. Library preparation and sequencing was performed according to manufacturer's recommendations. For each sample, a mean of 931 .000 reads per sample were generated. Data were normalized upon the manufacturer's recommendation to allow direct comparison between the different samples by the adjustments of number of reads. Limma, an R/Bioconductor software package, powered differential analyses for HTG Edge Sequencing analyses.
  • Blood collected in citrate containing tubes 2.7 mL was processed by separating cell-free plasma from blood cells within 15 minutes of collection by centrifugation at 1 ,500xg for 15 minutes. The supernatant plasma was transferred to a new tube. Tubes were kept at -70°C. The citrated plasma samples were used for the identification of new miRNA biomarkers with Small RNA Sequencing technology. To proceed to RNA extraction, plasma tubes were then centrifuged at 13,000xg for 2 min to pellet and remove the platelets. The supernatant platelet-free plasma was transferred to a new tube, frozen in liquid nitrogen and stored at -80°C.
  • RNA with preserved miRNAs was extracted from 400 ⁇ of platelet-free plasma by miRNeasy extraction kit (miRNeasy Serum/Plasma Kit (cat. no. 217184)) and using a plasma/QIAzol ratio of 1 :5 according to the manufacturer's instructions.
  • miRNeasy extraction kit miRNeasy Serum/Plasma Kit (cat. no. 217184)
  • plasma/QIAzol ratio 1 :5 according to the manufacturer's instructions.
  • Synthetic spiked-in C. elegans miR-39 was added to the samples [3,125 fmoles] prior to RNA extraction as internal control of RNA extraction process.
  • the elution was performed in 18 ⁇ of elution buffer. 15 ⁇ _ were used in the Small RNA Sequencing process.
  • Multiplex circ-microRNA sequencing library protocol V.I.G v2 is the protocol type and version used. Briefly, first, a 3' adenylated DNA adaptor is ligated to the total sample in the absence of ATP preventing the self-ligation of miRNAs. The RT primer complementary of the 3' adaptor is added at this stage with which it will form a duplex thereby reducing the ligation between adaptors. Then a 5' RNA adaptor is ligated in the presence of ATP at the other end of the miRNAs. A reverse transcription is therefore perfomed from the RT primer and finally these captured miRNAs are amplified by PCR with primers complementary to the 3' and 5' adaptors.
  • 70 differents barcodes (6 bp each) were used. 4 plates of 70 samples were processed, in two differents run. All the cDNA quantity was used in this pooling. Each pool of 10 cDNA was sized on an urea/acrylamide gel to select as usual the piRNA miRNA fraction of the libraries. The 70 samples of a 96 wells plate were sequenced on a unique Flow cell. Each pool of 10 libraries was sequenced on a unique flow cell lane, on single read 34bp + 7bp for the barcode discrimination using an Hiseq 2000.
  • a flow cell was composed by 7 lanes of 10 samples, in the 8th, a unique control and balanced library was sequenced. Each run was processed with a specific analysis. Each run is single read 34bp + 7bp, processed on the same Hiseq 2000.
  • the version of run software is HiSeq Control Software 2.0.12.0 and RTA 1 .17.213 which permits to insert a control lane on the flow cell to have a perfect base call. After the sequencing platform has generated the sequencing images, the data are analyzed in three steps: image analysis, base calling and bcl conversion.
  • This step consists to convert .bcl files into compressed FASTQ files.
  • Demultiplexing is done by CASAVA1 .8.2 (llumina software). It needs a BaseCalls directory and a sample sheet (.csv format) which stores information provided from flow cell content and separate multiplexed sequence runs by barcode. 0 mismatche is allowed in the barcode and only PF (Passing Filters) sequences are kept in FASTQ files.
  • Cutadapt software is used to trim a 3' adaptor, it handles the sequence file which contains adapter or not according the input of adaptor sequence.
  • FastQC aims to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing pipelines. It provides a modular set of analyses which you can use to give a quick impression of whether your data has any problems of which you should be aware before doing any further analysis.
  • the objective of the analyses is to discover biomarkers that can be related to the identification of NASH patients to be treated.
  • Patients to be treated are defined differently according to the different parts of the study.
  • TBT2 Before the comparison between TBT2 vs NTBT2, the samples, that have between 100 000 and 5 000 000 of reads aligned with miRNA and that have fifty% of values at least one condition (TBT2/NTBT2), were kept.
  • the comparison of differential miRNA expression was performed using Limma with Voom normalization (FC ⁇ 1 .5; p-value ⁇ 0.05).
  • TBT2 are defined as:
  • NAS NAFLD Activity Score
  • NAS is defined as the sum of the steatosis score, hepatocyte ballooning score and lobular inflammation grade
  • TaqMan miRNA qRT-PCR Assay TaqMan MicroRNA Reverse transcription Kit (Ref: 4366597, Applied Biosystems, Carlsbad, CA), TaqMan MicroRNA Assay 20X (Ref: 4440888, Applied Biosystems) and TaqMan Universal Master Mix II (Ref: 4440040, Applied Biosystems).
  • Reverse transcriptions were performed using a GeneAmp® PCR System 9700 thermal cycler (Ref: 200005, Applied Biosystems).
  • Quantitative PCRs were performed using a CFX96 TouchTM Real-Time PCR Detection System - C1000 - IVD certified, (185-5095 IVD, BioRad).
  • miRNA ID sequences of miRNA of interest and Taq Man assay ID.
  • Synthetic hsa-miRNA (Integrated DNA Technologies) was diluted at 3.125 fmol/mL and 5 ⁇ _ was used for reverse transcription concurrently with RNA extracted from serum samples. The product was serially diluted and PCR was performed on all samples (standards and serum- derived RNA). Standard curve was performed and used to convert Cq data in copies/ ⁇ -. The Cq Determination mode was Regression. Quantitation is expressed in copies/ ⁇ . of serum format. The supplier is IDT for the synthetic hsa-miRNA. RESULTS a) EXAMPLE 1 : miR-132
  • hepatic levels of 728 rat mature miRNA species were simultaneously measured in 48 liver samples fromthe preclinical CDAA rat model of NASH.
  • the inventors have identified, notably and surprisingly, mir-132 which was the miRNA the most overexpressed in liver of rats fed a choline-deficient, l-amino acid- defined (CDAA) diet alone or in combination with 1 % Cholesterol diet versus liver of rats fed a CSAA control diet (figure 1 ).
  • rno-miR-132-3p was 42-fold increased in CDAA fed group versus CSAA control diet and 52-fold in CDAA + Cholesterol fed group versus CSAA. Elafibranor prevents NASH and fibrosis development in this model of severe NASH with high penetration of advanced fibrosis and decreases rno-miR-132-3p significantly compared with CDAA + Cholesterol condition.
  • each individual score of histology (Fibrosis, Hepatocyte Balloning, Lobular Inflammation and Steatosis) is correlated with rno-miR-132- 3p.
  • Significant inductions of rno-miR-132-3p were also observed with Hepatocyte Ballooning and Lobular inflammation.
  • rno-miR-132-3p is very significantly associated with pathology progression, fibrosis, hepatocyte balloning, lobular inflammation and steatosis severity.
  • Table 1 Affymetrix miRNA results and modulation of hepatic rno-miR-132-3p expression with pathology progression and the severity of individual histological scores. Results are expressed as mean of fold change with an associated p value. To assess the diagnostic value of rno-miR-132-3p, the authors measured its circulating levels using the gold standard method for quantitation of oligonucleotides in PFP, RT-qPCR, using specific Taq Man miRNA assays.
  • Result can be resumed as follows: As shown in figure 2, exposure of Wistar rats to the CDAA + cholesterol diet for 1 1 weeks, results in more severe hepatocyte ballooning and more advanced fibrosis, as compared to the rats administered with the classical CDAA diet without cholesterol supplementation (figure 2A). In parallel, circulating rno-miR-132-3p concentration was significantly higher in CDAA + cholesterol group than in CSAA control group. CDAA induces a 5-fold induction of circulating rno-miR-132-3p and cholesterol supplementation which aggravated the CDAA- induced NASH, significantly increases this induction. rno-miR-132-3p levels are 18-fold increased in CDAA + cholesterol group versus CSAA control group (figure 2B).
  • Elafibranor significantly attenuates hepatic inflammation, ballooning and fibrosis with modest effects on steatosis in liver (figure 2A). This preventive effect observed in liver is correlated with a decrease of circulating rno-miR-132-3p levels (figure 2B).
  • TBT refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT subject differs from a TBT subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT To Be Treated
  • NTBT Not-To-Be-Treated
  • Table 2 RT-qPCR experiments for confirmation/validation of overexpression of hsa-miR- 132-3p in To Be Treated (TBT) Patients versus Not-To-Be-Treated (NTBT) Patients.
  • TBT vs NTBT was calculated using the non-parametric Mann Whitney test.
  • TBT refers to patients with NAS ⁇ 4 with steatosis, hepatocyte ballooning and lobular inflammation scores ⁇ 1 and fibrosis stage ⁇ 2 at histological examination of a liver biopsy.
  • AUC Area under the curve of Receiver Operating Characteristic were obtained for identification of TBT vs NTBT.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death. From this analysis, the inventors have identified mir-125 which was commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, OBESE and RESOLVE-IT cohorts at inclusion.
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-125b-5p was significantly higher in TBT2 patients than in NTBT2 patients. These results were confirmed in the independent cohorts OBESE and RESOLVE-IT between TBT2 and NTBT2 patients.
  • hsa-miR-125b-5p serum concentrations were significantly higher in NTBT2 (NAFLD patients with minimal histological lesions) than in serum from HEALTHY subjects.
  • hsa-mir-125b-5p and hsa-miR-125b-2-3p were then measured using the gold standard method for quantitation of oligonucleotides in body fluids, RT-qPCR, using specific Taq Man miRNA assays. Result can be resumed as follows:
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte Ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-125b-5p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • hsa-mir-125b-5p serum concentrations were significantly higher in patients with significant fibrosis or higher fibrosis stage (F > 2) than in patients with no or minimal fibrosis (F ⁇ 2).
  • circulating level of hsa-miR-125b-5p and hsa-miR- 125b-2-3p positively correlated with steatosis score, lobular inflammation score, hepatocyte ballooning score. Consequently, circulating levels of miR-125b-5p and hsa-miR-125b-2-3p significantly and positively correlated with NAS and activity Index. Finally, there was a strong correlation between circulating level of miR-125b-5p and fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death. From this analysis, the inventors have identified mir-505 which was commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, OBESE and RESOLVE-IT cohorts at inclusion.
  • the number of reads per million for hsa-miR-505-3p was significantly higher in TBT2 patients than in NTBT2 patients.
  • 181 RPM Reads per million
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-505-3p was significantly higher in TBT2 patients than in NTBT2 patients.
  • hsa-miR-505-3p serum concentrations were significantly higher in NTBT2 (NAFLD patients with minimal histological lesions) than in serum from healthy subjects.
  • level of hsa-mir-505-3p was then measured using the gold standard method for quantitation of oligonucleotides in body fluids, RT-qPCR, using specific Taq Man miRNA assays. Result can be resumed as follows: As shown in table 4 and figures 1 1 -12-13-14, in GOLDEN-DIAG and RESOLVE-IT at inclusion and in GOLDEN-DIAG one year later, hsa-mir-505-3p serum concentration was significantly higher in TBT2 patients than in NTBT2 patients.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte Ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-505-3p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • circulating level of hsa-miR-505-3p positively correlated with steatosis score, lobular inflammation score, hepatocyte ballooning score. Consequently, circulating level of miR-505-3p significantly and positively correlated with NAS and activity Index. Finally, there was a strong correlation between circulating level of miR-505-3p and fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death. From this analysis, the inventors have identified mir-365 which was commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, OBESE and RESOLVE-IT cohorts at inclusion.
  • the number of reads per million for hsa-miR-365a-3p was significantly higher in TBT2 patients than in NTBT2 patients.
  • 413 RPM (Reads per million) were obtained in TBT2 patients versus 276 RPM for NTBT2 patients for hsa-miR-365a-3p in Golden Diag at Inclusion.
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-365a-3p was significantly higher in TBT2 patients than in NTBT2 patients. These results were confirmed in the independent cohorts OBESE and RESOLVE-IT between TBT2 and NTBT2 patients.
  • hsa-miR-365a-3p serum concentrations were significantly higher in NTBT2 (NAFLD patients with minimal histological lesions) than in serum from healthy subjects.
  • level of hsa-mir-365a-3p was then measured using the gold standard method for quantitation of oligonucleotides in body fluids, RT-qPCR, using specific Taq Man miRNA assays. Result can be resumed as follows:
  • hsa-mir-365a-3p serum concentration was significantly higher in TBT2 patients than in NTBT2 patients.
  • hsa-miR-365a-3p serum concentration was higher in TBT2 patients than in NTBT2 patients with a trend toward significance.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-365a-3p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • circulating level of hsa-miR-365a-3p positively correlated with steatosis score, lobular inflammation score, hepatocyte ballooning score. Consequently, circulating level of miR-365a-3p significantly and positively correlated with NAS and activity Index. Finally, there was a strong correlation between circulating level of miR-365a-3p and fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death.
  • miR-22 which was commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, OBESE and RESOLVE-IT cohorts at inclusion.
  • the number of reads per million for hsa-miR-22-5p, and hsa-miR-22-3p were significantly higher in TBT2 patients than in NTBT2 patients.
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-22-5p and hsa-miR-22-3p were significantly higher in TBT2 patients than in NTBT2 patients.
  • level of hsa-mir-22-3p was then measured using the gold standard method for quantitation of oligonucleotides in body fluids, RT-qPCR, using specific Taq Man miRNA assays. Result can be resumed as follows:
  • hsa-mir-22-3p serum concentration was significantly higher in TBT2 patients than in NTBT2 patients.
  • hsa-miR- 22-3p serum concentration was higher in TBT2 patients than in NTBT2 patients with a trend toward significance in OBESE.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-22-3p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non- NASH and mild NASH patients (NAS ⁇ 4).
  • NAS ⁇ 4 Active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 hsa-miR-22-3p serum concentration is higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4) with a trend toward significance.
  • hsa-miR-22-3p serum concentration is higher in patients with significant fibrosis or higher fibrosis stage (F > 2) than in patients with no or minimal fibrosis (F ⁇ 2) with a trend toward significance.
  • TBT2 definition NAS > 4 and F > 2, and at least one point in steatosis, lobular inflammation and hepatocyte ballooning scores
  • NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death.
  • mir-378 which was commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, OBESE and RESOLVE-IT cohorts at inclusion.
  • the number of reads per million for hsa-miR-378a-3p, hsa-miR-378d, hsa-miR-378e, hsa-miR-378f, hsa- miR-378g, and hsa-miR-378i were significantly higher in TBT2 patients than in NTBT2 patients.
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-378a-3p, hsa-miR-378d, hsa- miR-378e, hsa-miR-378f, hsa-miR-378g, and hsa-miR-378i, were significantly higher in TBT2 patients than in NTBT2 patients.
  • level of hsa-mir-378a-3p was then measured using the gold standard method for quantitation of oligonucleotides in body fluids, RT-qPCR, using specific Taq Man miRNA assays. Result can be resumed as follows:
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte Ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-378a- 3p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non- NASH and mild NASH patients (NAS ⁇ 4).
  • hsa-mir- 378a-3p serum concentrations were significantly higher in patients with significant fibrosis or higher fibrosis stage (F > 2) than in patients with no or minimal fibrosis (F ⁇ 2).
  • TBT2 definition NAS > 4 and F > 2, and at least one point in steatosis, lobular inflammation and hepatocyte ballooning scores
  • NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death.
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-320e-3p were higher in TBT2 patients than in NTBT2 patients.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-320e- 3p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • NAS ⁇ 4 Active-NASH
  • RESOLVE-IT hsa- miR-320e-3p serum concentration is higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non- NASH and mild NASH patients (NAS ⁇ 4) with a trend toward significance.
  • TBT2 definition NAS > 4 and F > 2, and at least one point in steatosis, lobular inflammation and hepatocyte ballooning scores
  • NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death.
  • the inventors also used liver biopsies and serum samples collected at the end of the one- year treatment period of GOLDEN trial as a third independent data set and once again confirmed that the number of reads per million for hsa-miR-885-5p were significantly higher in TBT2 patients than in NTBT2 patients.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-885-5p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non- NASH and mild NASH patients (NAS ⁇ 4).
  • NAS ⁇ 4 Active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 in GOLDEN Diag
  • hsa- miR-885-5p serum concentration is higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non- NASH and mild NASH patients (NAS ⁇ 4) with a trend toward significance.
  • TBT2 definition NAS > 4 and F > 2, and at least one point in steatosis, lobular inflammation and hepatocyte ballooning scores
  • NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death.
  • the inventors have identified miR-483 which was commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, and OBESE cohorts at inclusion. As shown in the table 3, notably and surprisingly, in GOLDEN-DIAG cohort at inclusion, the number of reads per million for hsa-miR-483-5p, were significantly higher in TBT2 patients than in NTBT2 patients.
  • hsa-miR-483-5p 15017 RPM (Reads per million) were obtained in TBT2 patients versus 6230 RPM for NTBT2 patients for hsa-miR-483-5p in Golden Diag at Inclusion.
  • level of hsa-mir-483-5p was then measured using the gold standard method for quantitation of oligonucleotides in body fluids, RT-qPCR, using specific Taq Man miRNA assays. Result can be resumed as follows: As shown in table 4 and figures 39-40-41 , in GOLDEN-DIAG, in GOLDEN-DIAG one year later, and in OBESE, hsa-miR-483-5p serum concentration was significantly higher in TBT2 patients than in NTBT2 patients.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-483-5p serum concentrations were significantly higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • NAS ⁇ 4 Active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 active-NASH
  • NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning
  • results presented in the following table 5 illustrate significant correlations between changes in circulating levels of hsa-miR-483-5 and evolution of NAFLD Activity Score and Activity Index after 52 weeks in GOLDEN patients.
  • these results based on measurement of levels of miRNA in serum and plasma samples using two different methodologies (Small RNA Sequencing and RTqPCR) support the use of hsa-miR-483-5p and more generally hsa-miR-483 related oligonucletotides as circulating diagnostic biomarkers for identification of patients with NAFLD (NAS ⁇ 1 ), NASH (NAS > 3 with at least 1 point in steatosis, at least 1 point in lobular inflammation and at least 1 point in hepatocyte ballooning scores), Active-NASH (NAS > 4 with at least 1 point in steatosis, at least 1 point in lobular inflammation and at least 1 point in hepatocyte ballooning scores), significant fibrosis (F > 2), and/or Active-NASH and
  • TBT2 definition NAS ⁇ 4 and F ⁇ 2, and at least one point in steatosis, lobular inflammation and hepatocyte ballooning scores
  • NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT2 patients should be treated for their increased risk of evolution to serious liver outcomes like cirrhosis, HCC, liver failure, liver transplant and liver death.
  • hsa-miR-30a-5p hsa-miR-422a-5p
  • hsa- miR-100-5p hsa-miR-4324-3p
  • hsa-miR-4324-3p which were significantly and commonly overexpressed in serum samples of TBT2 patients in comparison to NTBT2 patients in GOLDEN-DIAG, at inclusion and one year later.
  • TBT1 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT1 subject differs from a TBT1 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • TBT7 vs. NTBT7 As shown in figures 43-46 when applying a third definition of TBT patients and NTBT patients (TBT7 vs. NTBT7) in the GOLDEN-DIAG cohort at inclusion, analyses showed that hsa-miR-30, hsa-miR-422a-5p, hsa-miR-100, and hsa-miR-4324-3p serum concentrations were significantly higher in TBT7 patients than in NTBT7 patients.
  • TBT7 refers to patients with NAS ⁇ 4 with at least 1 point in steatosis, hepatocyte ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 1 at histological examination of a liver biopsy.
  • a NTBT7 subject differs from a TBT7 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage.
  • hsa-miR-30a-5p serum concentrations were higher in patients with significant fibrosis or higher fibrosis stage (F ⁇ 2) than in patients with no or minimal fibrosis (F ⁇ 2).
  • Hsa-miR-30a-5p serum concentrations were also higher in patients with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • hsa- miR-100-5p serum concentrations were higher in patients with significant fibrosis or higher fibrosis stage (F ⁇ 2).
  • Hsa-miR-100-5p serum concentrations were also higher with Active- NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • hsa- miR-422a-5p serum concentrations were significantly higher in patients with significant fibrosis or higher fibrosis stage (F ⁇ 2).
  • High levels of hsa-miR-422a-5p serum concentrations were also associated with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) compared to non-NASH and mild NASH patients (NAS ⁇ 4).
  • hsa- miR-4324 serum concentrations were significantly higher in patients with significant fibrosis or higher fibrosis stage (F ⁇ 2).
  • Hsa-miR-4324-3p serum concentrations were also significantly associated with Active-NASH (NAS ⁇ 4 with at least one point in steatosis, lobular inflammation and hepatocyte ballooning) than in non-NASH and mild NASH patients (NAS ⁇ 4).
  • circulating level of hsa-miR-30a-5p, hsa-miR-100-5p, hsa- miR-422a-5p, and hsa-miR-4324-3p positively correlated with lobular inflammation score and hepatocyte ballooning score in GOLDEN-DIAG study. Consequently, circulating level of hsa- miR-30a-5p, hsa-miR-100-5p, hsa-miR-422a-5p, and hsa-miR-4324-3p correlated with NAS and with activity index.
  • hsa_miRNA RPM in NTBT2 RPM in TBT2 Fold Change p value hsa-mi -125b-5p 650 861 1,37 2,94E-05 hsa-miR-505-3p 150 181 1,2 2,80E-03 hsa-miR-365a-3p 31 44 1,51 l,38E-03 hsa-miR-22-5p 403 473 1,17 2,69E-02 hsa-miR-22-3p 24069 29286 1,19 8,llE-04 hsa-miR-378a-3p 242 283 1,21 4,28E-04 hsa-miR-378d 97 121 1,26 7,48E-05 hsa-miR-378e 80 105 1,31 2,32E-06 hsa-miR-378f 140 168 1,2 l,99E-04 hsa-miR-378g 153 178 1,15 4,80E-03 hs
  • hsa_miRNA RPM in NTBT2 RPM in TBT2 Fold Change p value hsa-miR-125b-5p 211 344 1,4 7,21E-04 hsa-miR-505-3p 96 115 1,18 2,63E-02 hsa-miR-365a-3p 19 31 1,32 9,67E-02 hsa-miR-22-5p 245 290 1,17 8,24E-02 hsa-miR-22-3p 16268 19662 1,23 l,12E-03 hsa-miR-378a-3p 154 179 1,16 8,68E-03 hsa-miR-378d 61 78 1,25 9,31E-04 hsa-miR-378e 71 86 1,17 2,13E-02 hsa-miR-378f 109 113 1,06 3,10E-01 hsa-miR-378g 138 144 1,05 4,75E-01 hsa
  • hsa_miRNA RPM in NTBT2 RPM in TBT2 Fold Change p value hsa-mi -125b-5p 468 587 1,29 l,60E-03 hsa-miR-505-3p 159 177 1,14 2,63E-02 hsa-miR-365a-3p 25 33 1,08 5,92E-01 hsa-miR-22-5p 372 396 1,07 3,33E-01 hsa-miR-22-3p 22255 26903 1,2 3,90E-04 hsa-miR-378a-3p 194 236 1,23 l,25E-04 hsa-miR-378d 85 105 1,24 7,28E-04 hsa-miR-378e 85 109 1,3 9,01E-06 hsa-miR-378f 127 151 1,2 2,23E-04 hsa-miR-378g 143 173 1,22 5,llE-05 hsa-m
  • Table 3 HGT-Edge- Sequencing experiments and number of reads per millions (RPM) obtained for hsa-miR-125b-5p in To-Be-Treated (TBT2) versus Not-To-Be-Treated (NTBT2) patients.
  • Reads per million are expressed as mean of NTBT2 and TBT2 patient groups (Golden Diag Study - At Inclusion (109 TBT2 and 161 NTBT2 patients) and Golden Diag - One Year Later (76 TBT2 and 147 NTBT2); Obese (50 TBT2 and 202 NTBT2 patients); RESOLVE-IT (87 TBT2 and 90 NTBT2) respectively;
  • TBT2 refers to patients with NAS ⁇ 4 with at least 1 point in Steatosis, Hepatocyte Ballooning and Lobular Inflammation scores and fibrosis stage ⁇ 2 at histological examination of a liver biopsy.
  • NTBT2 subject differs from a TBT2 subject in at least one point lesser grade in steatosis, hepatocyte ballooning, lobular inflammation scores, NAS and/or fibrosis stage. Healthy subjects are 100 EFS subjects without medication.
  • TBT2 To Be Treated
  • NTBT2 Not-To-Be-Treated
  • Table 4 RT-qPCR experiments for confirmation/validation of overexpression of hsa-miRNAs in To Be Treated (TBT2) Patients versus Not-To-Be-Treated (NTBT2) Patients.
  • TBT2 vs NTBT2 was calculated using the non-parametric Mann Whitney test.
  • TBT2 refers to patients with NAS ⁇ 4 with steatosis, hepatocyte ballooning and lobular inflammation scores ⁇ 1 and fibrosis stage ⁇ 2 at histological examination of a liver biopsy.
  • AUC Area Under the Curve of Receiver Operating Characteristic were obtained for identification of TBT2 vs NTBT2.
  • Table 5 Correlation of changes in serum levels of hsa-miRNAs and the evolutions of NAS, Activity Index, and Liver Fibrosis during the one-year GOLDEN trial. References
  • miChip a microarray platform for expression profiling of microRNAs based on locked nucleic acid (LNA) oligonucleotide capture probes.
  • LNA locked nucleic acid
  • RNA 12 187-191 Satake E, Pezzolesi MG, Md Dom Zl, Smiles AM, Niewczas MA, Krolewski AS (2016) Circulating miRNA Profiles Associated With Hyperglycemia in Patients With Type 1 Diabetes. Diabetes 67: 1013-1023

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Abstract

La présente invention concerne une nouvelle méthode non invasive pour le diagnostic d'une maladie du foie gras non alcoolique, en particulier de la stéatohépatite non alcoolique et/ou de la fibrose hépatique.
PCT/EP2018/074977 2017-09-15 2018-09-14 Diagnostic non invasif de maladies du foie gras non alcooliques, de la stéatohépatite non alcoolique et/ou de la fibrose hépatique Ceased WO2019053235A1 (fr)

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