Welcome to our new mini-series about liver organoids. In Part 1, we focus on non-alcoholic steatohepatitis (NASH), a severe and prevalent form of non-alcoholic fatty liver disease for which no drugs have been approved. We look at the current treatment landscape for NASH, some ongoing efforts to develop new treatments, and provide a brief overview of current 2D cellular models for NASH and their limitations. Finally, we touch on liver organoids as a robust and promising model to advance new treatments for NASH.
Non-alcoholic steatohepatitis, or NASH, is the most advanced and severe form of non-alcoholic fatty liver disease (NAFLD), which is characterized by a buildup of lipids (hepatic steatosis), hepatocyte injury, and swelling and inflammation of the liver. In contrast to other fatty liver diseases, NASH is not caused by excessive alcohol consumption or side effects of medication, but is instead most often brought on by lifestyle choices, e.g., chronic high sugar and high fat intake and lack of exercise.
NASH is most prevalent in individuals who are overweight or obese. Other prominent risk factors include type 2 diabetes, high cholesterol, metabolic disorders, thyroid disorders, polycystic ovarian syndrome and sleep apnea.
NASH is often described as a silent killer; symptoms tend to be mild and non-specific in the early stages, e.g., fatigue or mild pain in the upper right abdomen, which means that individuals can have the disease for many years without knowing it. As the condition progresses, symptoms of cirrhosis may appear, including bruising, itchy and yellowing of the skin (jaundice), fluid buildup in the abdomen, swelling, confusion, drowsiness and others.
Undiagnosed NASH leads to serious and potentially fatal conditions, including advanced fibrosis, cirrhosis, liver failure, and primary liver cancer, as well as consequences beyond the liver, for instance cardiovascular complications. More than 20% of NASH patients develop cirrhosis within a decade of diagnosis and, compared to the general population, have a ten-fold greater risk of liver-related mortality.
The global NASH burden is increasing rapidly, but treatment options are poor
According to the American Liver Foundation, NAFLD is the most common chronic liver condition in the U.S., and is estimated to affect approx. 25 % of all adults. Of those with NAFLD, about 20 % are estimated to have NASH (this equates to approx. 5% of adults in the U.S. with NASH). The reason not everyone with NAFLD develops NASH is unknown, but research suggests that certain genes and environmental factors may play a role.
As the impact of risk factors intensifies worldwide, e.g., the increasing burden of type 2 diabetes and obesity, the prevalence of NASH is also rising steadily. Forecasts made in recent years predict that NASH prevalence will increase by 63% between 2015 and 2030, and that NASH is expected to become the leading cause of liver transplantation in the U.S. between 2020-2025.
Current treatment approaches to NASH include lifestyle changes, e.g., sustained weight loss, dietary adjustments and exercise regimes, as well as the use of certain medications to control co-existing diseases where present, e.g., diabetic medications and cholesterol-lowering agents. Liver transplantation may be an option in severe cases where liver failure is imminent; however transplantation surgery is not without serious risks, and donor livers are not available for everyone who needs them.
Despite intense efforts, no therapy has yet been approved for NASH (as of March 2023)
NASH is a complex and multifaceted disease that occurs along a continuum of stages involving metabolic dysfunction, inflammation and fibrosis. Drug development efforts to date have focused primarily on treating late stages of disease but treating earlier stages could impact a greater number of individuals as a preventative measure.
A number of candidates designed to inhibit fibrosis and inflammation in NASH have made it to late-stage clinical trials, but these have either failed or are yet not approved. For instance, the small molecule PPAR agonist Elafibranor (developed by Genfit, France), which was designed to treat NASH regardless of severity was granted Fast Track Designation by the FDA in 2014, but was later pulled by Genfit at Phase 3 in 2020 due to lack of efficacy. Another French company, Inventiva, is developing a pan-PPAR agonist Lanifibranor that is designed to modulate all components of NASH (metabolism, steatosis, inflammation and fibrosis). Lanifibranol demonstrated safety and efficacy in Phase 1 and 2 studies, and is currently being evaluated for efficacy and long-term safety in a pivotal Phase 3 study.
U.S.-based Intercept Pharmaceuticals is edging closer to a potential FDA approval for its NASH candidate, obeticholic acid, a farnesoid X receptor agonist with anti-cholestatic and hepato-protective properties, which is currently sold under the brand name Ocaliva as a treatment for another liver disease, primary biliary cholangitis. Intercept’s first application for marketing authorization was rejected by the FDA in 2020, but following a change to the Phase 3 study endpoint and a longer safety follow-up, the FDA accepted Intercept’s second application and set a target decision date in June 2023 following a one-day expert hearing to take place in May 2023. Clinical data released so far indicates that Ocaliva may benefit individuals with pre-cirrhotic NASH by halting progression, but is unlikely to benefit late-stage NASH patients with severe cirrhosis.
Another strong contender in this space is Madrigal Pharmaceuticals (U.S.), whose liver-directed selective thyroid hormone receptor agonist, resmetirom, achieved the liver histological improvement endpoints that the FDA had proposed as reasonably likely to predict clinical benefit to support accelerated approval for the treatment of NASH with liver fibrosis. Resmetiron is being evaluated in the Phase 3 Maestro-NASH trial, and when the most recent clinical data was released in December, resmetirom became the first therapeutic for NASH to show improvements on liver scarring and NASH resolution compared to placebo.
Earlier this week, U.S.-headquartered company 89bio announced positive topline data from the Phase 2b ENLIVEN trial evaluating treatment with pegozafermin in patients with NASH. Two of the three doses tested in this study met the primary histology endpoints with statistical significance, as per the FDA’s guidance on endpoints and statistical analysis. Pegozafermin is an engineered glycoPEGylated analog of fibroblast growth factor 21 (FGF21). FGF21 is an endogenous hormone normally produced in the liver at low levels, which modulates important drivers of lipid metabolism and NASH including triglyceride reduction, glycemic control, steatosis, inflammation and fibrosis.
Another U.S. company Akero Therapeutics announced late in 2023 that it had completed enrollment of the Phase 2b SYMMETRY main study to evaluate the potential of efruxifermin (EFX) to slow or reverse progression of cirrhosis in NASH patients with compensated cirrhosis fibrosis stage 4. Histology data from a Phase 2a proof-of-concept study in patients with cirrhosis due to NASH were promising, and the company expects to release clinical data from SYMMETRY later this year. EFX is a fusion protein that is engineered to mimic the biological activity profile of FGF21, to reduce liver fat and inflammation, reverse fibrosis, increase insulin sensitivity and improve lipoproteins.
The cells behind NASH and the limitations of 2-dimensional disease models
The massively increasing burden of NASH on healthcare has greatly motivated efforts in recent years to identify drug targets and develop new therapies, some of which are described above. Despite great strides in our understanding of the etiology and disease mechanisms underlying NASH, the development of effective anti-fibrotic therapies has been greatly hampered by the limitations of existing pre-clinical models, the lack of validated translational models and poor predictability of animal models.
Research findings in recent years suggest that crosstalk between multiple cell types plays a pivotal role in NASH disease progression. Liver sinusoidal endothelial cells (LSEC)s, Kupffer cells, and stellate cells (HSCs) are among the most abundant non-parenchymal cell (NPC) types distributed in hepatic sinusoids (the small blood vessels situated between the rows of hepatocytes), which collectively constitute 35 % of the total cell number in the liver.
LSECs play a critical role in maintaining liver homeostasis through their interactions with hepatocytes, lymphocytes, neutrophils, macrophages, and hepatic stellate cells. LSECS can trigger steatosis, inflammatory responses, and fibrogenesis through communicating with surrounding cells, and are often referred to as the gatekeeper of the hepatic microenvironment, since they constitute the physical interface between the circulatory system and the sinusoids. Kupffer cells, which are hepatic macrophages, are the key cells regulating liver immunity. These become activated upon liver damage to release inflammatory cytokines and chemokines, and together with surrounding cells they participate in the inflammatory response, fibrogenesis and vascular remodeling in NASH. Hepatic stellate cells (HSCs) are the primary extracellular matrix–producing cells in the liver, and when activated they generate a temporary scar at the site of injury to protect the liver from further damage. However, during chronic liver disease, prolonged and repeated activation of stellate cells causes liver fibrosis. In NASH, HSCs differentiate into myofibroblast-like cells that cause fibrosis, which increases a patient’s risk of developing cirrhosis and liver cancer.
The most widely used 2D cellular models to study NASH are based on in vitro mono- or co-cultures of cells growing on a flat surface, e.g., a hepatocyte monoculture system in which primary hepatocytes are plated on 2D collagen-coated dishes, hepatic stellate cell monocultures in various matrices with varying tensile strength, and Kupffer cell monocultures for the measurement of inflammatory responses. Currently used 2D co-culture models for NASH include cultures of hepatocytes with stromal fibroblasts, or Kupffer cells co-cultured with stellate cells.
2D cellular models for NASH are limited in their usefulness and relevance to NASH physiology because of their short-lived liver-specific functionality and responses and poor cellular longevity, which preclude long-term and repeated dosing studies to screen new therapeutic candidates. Furthermore, their flat nature and limited cell types prevents the development of critical interactions between hepatocytes and other cell types that may be involved in manifesting NASH pathobiology.
3D liver organoid models are key to advancing new therapies for NASH
A physiologically-relevant human NASH model should recapitulate the important (and therapeutically-targeted) aspects of the disease in vitro, and resemble the functionality and morphology of the human liver as much as possible.
Liver organoids, which are 3D spheroids composed of multiple cell types, generated from liver progenitor cells or reprogrammed from pluripotent stem cells, are a highly attractive model to study NASH and screen new candidate therapies. With greater longevity than 2D cell models in culture, spatial and temporal liver-relevant gene expression, the propensity for cell-cell communication and physical cell-cell interactions, and amenability to genetic manipulation, liver organoids allow for a much more complex and diverse experimental strategy than any 2D model.
Stay tuned for Part 2 of this series, where we will take a closer look at how liver organoids may be used to study NASH biology and aid the development of new therapies.
References:
- Mukherjee S, Zhelnin L, Sanfiz A, Pan J, Li Z, Yarde M, McCarty J, Jarai G. Development and validation of an in vitro 3D model of NASH with severe fibrotic phenotype. Am J Transl Res. 2019 Mar 15;11(3):1531-1540. PMID: 30972180.
- Afonso MB, Rodrigues PM, Carvalho T, Caridade M, Borralho P, Cortez-Pinto H, Castro RE, Rodrigues CM. Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis. Clin Sci (Lond). 2015 Oct 1;129(8):721-39. doi: 10.1042/CS20140732. doi: 10.1042/CS20140732.
- Hendriks D, Brouwers JF, Hamer K, Geurts MH, Luciana L, Massalini S, López-Iglesias C, Peters PJ, Rodríguez-Colman MJ, Chuva de Sousa Lopes S, Artegiani B, Clevers H. Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis. Nat Biotechnol. 2023 doi: 10.1038/s41587-023-01680-4. PMID: 36823355.
- Estes C, Razavi H, Loomba R, et al. Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease. Hepatology. 2018;67(1):123-133. doi: 10.1002/hep.29466.
- Wong RJ, Aguilar M, Cheung R, et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology. 2015;148:547-555. doi: 10.1002/hep.29466.
- Du W, Wang L. The Crosstalk Between Liver Sinusoidal Endothelial Cells and Hepatic Microenvironment in NASH Related Liver Fibrosis. Front Immunol. 2022 Jun 28;13:936196. doi: 10.3389/fimmu.2022.936196.
Karen O’Hanlon Cohrt is an independent Science Writer with a PhD in biotechnology from Maynooth University, Ireland (2011). After her PhD, Karen relocated to Denmark where she held postdoctoral positions in mycology and later in human cell cycle regulation, before moving to the world of drug discovery. Karen has been a full-time science writer since 2017, and has since then held numerous contract roles in science communication and editing spanning diverse topics including diagnostics, molecular biology, and gene therapy. Her broad research background provides the technical know-how to support scientists in diverse areas, and this in combination with her passion for learning helps her to keep abreast of exciting research developments as they unfold. Karen is currently based in Ireland, and you can follow her on Linkedin here.
