Human Liver ORganoids as a Model to Study the Development of Non-Alcoholic SteatOhepatitis (NASH) (REASON)

Human Liver ORganoids as a Model to Study the Role of the I148M Variant of the PNPLA3 Gene in the Development of Non-Alcoholic SteatOhepatitis (NASH)

The primary objective of the study is to generate and characterize three-dimensional models, called "assembloids", composed of the main liver cell populations (in particular from the co-culture of organoids with stellate cells, responsible for fibrogenesis, deriving from clinical samples). These models will be used in order to imitate the first phases of the onset of steatohepatitis, in conditions of altered lipid metabolism (induced through exposure to the main environmental determinants of this condition: excess fatty acids, fructose, cholesterol) in the presence or absence of the mutation I148M of PNPLA3. Other genetic variants will also be analyzed, such as TM6SF2, MBOAT7 and GCKR, which have previously been correlated with the development of non-alcoholic steatohepatitis.

Further objectives will be: 1) identify new biomarkers of pathological activation of human stellate cells and progression of liver damage, to be subsequently validated in clinical case series for future use in clinical management for individual risk stratification; 2) study the epigenetic factors that underlie the onset of non-alcoholic steatohepatitis and its progression to fibrosis, cirrhosis and HCC; 3) evaluate the impact of antisense oligonucleotides directed against PNPLA3 on the severity of the "steatohepatitic" phenotype (lipid accumulation, lipotoxicity and inflammation and fibrogenesis) in assembloids

Study Overview

• Status: Recruiting
• Conditions: NAFLD
• Intervention / Treatment: Genetic: Liver resection to isolate cells

Detailed Description

Non-alcoholic fatty liver disease (NAFLD) represents the main cause of liver damage and is found in approximately one third of the population (25-30%). NAFLD encompasses a broad spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). NASH is found in 20-30% of patients with NAFLD and can progress to cirrhosis and hepatocellular carcinoma.

Progress recorded in recent years in the field of genetics has highlighted how genetic factors play a key role in the susceptibility, severity and long-term prognosis of the disease. In particular, in a genome-wide association study conducted on a multi-ethnic population, the PNPLA3 gene was identified as the main genetic factor that is strongly associated with intra-hepatic fat content, independently of body mass and insulin resistance.

The primary objective of the study is to generate and characterize three-dimensional models, called "assembloids", composed of the main liver cell populations (in particular from the co-culture of organoids with stellate cells, responsible for fibrogenesis, deriving from clinical samples). These models will be used in order to imitate the first phases of the onset of steatohepatitis, in conditions of altered lipid metabolism (induced through exposure to the main environmental determinants of this condition: excess fatty acids, fructose, cholesterol) in the presence or absence of the mutation I148M of PNPLA3. Other genetic variants will also be analyzed, such as TM6SF2, MBOAT7 and GCKR, which have previously been correlated with the development of non-alcoholic steatohepatitis.

Further objectives will be: 1) identify new biomarkers of pathological activation of human stellate cells and progression of liver damage, to be subsequently validated in clinical case series for future use in clinical management for individual risk stratification; 2) study the epigenetic factors that underlie the onset of non-alcoholic steatohepatitis and its progression to fibrosis, cirrhosis and HCC; 3) evaluate the impact of antisense oligonucleotides directed against PNPLA3 on the severity of the "steatohepatitic" phenotype (lipid accumulation, lipotoxicity and inflammation and fibrogenesis) in assembloids

• Study Type: Interventional
• Enrollment (Estimated): 60
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Luca Vittorio Carlo Valenti; Phone Number: 56595 02 5503 6595; Email: luca.valenti@policlinico.mi.it

Study Locations

• Italy
  • Milano
    • Milan, Milano, Italy, 20122
      • Recruiting
      • Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico - Istituto di Ricovero e Cura a Carattere Scientifico di natura pubblica
      • Contact: Luca Vittorio Carlo Valenti; Phone Number: 56595 02 5503 6595; Email: luca.valenti@policlinico.mi.it

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Adult patients who have given consent to participate in the study and listed for the following procedures will be included:

• liver biopsy for suspected non-alcoholic steatohepatitis (NASH) at the time of diagnosis;
• liver resection for hepatocarcinoma or other liver lesions (including secondaries from other neoplasms and benign focal lesions, which will allow obtaining healthy starting liver tissue);
• post-transplant healthy liver biopsies;
• cholecystectomies.

It will also be required:

• availability to sign informed consent for the study
• availability of DNA sample for genetic analysis and clinical data,
• blood sampling for genetic and epigenetic analyzes and analysis of non-coding RNAs (lncRNAs, miRNAs and circRNAs).

Patients will be excluded who present:

• positivity for chronic viral hepatitis (HCV-RNA and/or HBsAg);
• positivity to other liver diseases such as autoimmune and viral hepatitis (hepatitis B and C), hereditary hemochromatosis, alpha-1-antitrypsin deficiency, Wilson's disease.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Liver resection to isolate cells; Isolation and culture of organoids Isolation of hepatic stellate cells Isolation and culture of sinusoidal stellate cells Generation of assembloids	Genetic: Liver resection to isolate cells; Isolation and culture of organoids Isolation of hepatic stellate cells Isolation and culture of sinusoidal stellate cells Generation of assembloids

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Generation of treatment-related assembloids	Isolation of epithelial cells to impact assessment of the I148M variant on the steatohepatic phenotype in human liver assembloids and analysis of the variants of the TM6SF2, MBOAT7 and GCKR genes previously related to the development of non-alcoholic steatohepatitis	up to 35 months
Rate of PNPLA3 p.I148M variant in steatohepatitis development through human liver organoids	Knowledge of the gene expression of lipotoxicity and expression of inflammatory markers;; Knowledge of the molecular mechanisms to evaluate the state of activation of star cells, the metabolism of retinol and the deposition of extracellular matrix and estimate	up to 35 months

Collaborators and Investigators

• Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

Study record dates

Study Major Dates

• Study Start (Actual): July 1, 2021
• Primary Completion (Estimated): July 31, 2030
• Study Completion (Estimated): July 31, 2032

Study Registration Dates

• First Submitted: March 8, 2024
• First Submitted That Met QC Criteria: February 25, 2025
• First Posted (Actual): March 4, 2025

Study Record Updates

• Last Update Posted (Actual): November 20, 2025
• Last Update Submitted That Met QC Criteria: November 17, 2025
• Last Verified: November 1, 2025

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Digestive System Diseases; Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease
• Other Study ID Numbers: REASON

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No