Research consortium aims to discover RNA medicine for treating an emerging liver disease
Nonalcoholic steatohepatitis (NASH) affects millions of people worldwide. However, there are currently no approved therapies for treating NASH. An international research team of scientists from Denmark and the United States will focus on discovering microRNA-targeted therapeutics to tackle NASH.
Nonalcoholic steatohepatitis (NASH) is a widespread, chronic liver disease affecting millions of people worldwide. Most people with NASH are asymptomatic and, thus, the disease can quietly progress to a life-threatening condition.
Although the global prevalence of NASH is rapidly increasing, there are currently no approved therapies for NASH. As a result, several biotech and major pharmaceutical companies have jumped into the NASH field to develop drugs for treating NASH.
In addition, researchers around the world, including Sakari Kauppinen from the Center for RNA Medicine at Aalborg University, are working hard to develop new therapeutic strategies to treat NASH.
“New therapies for NASH are urgently needed, since many patients with NASH are at risk of developing cirrhosis and liver cancer. Our goal is to discover microRNA-based drugs and to assess their therapeutic potential to tackle NASH,” says Sakari Kauppinen.
In 2018, the Novo Nordisk Foundation awarded a Challenge Programme grant of DKK 60 million to an international research consortium led by Sakari Kauppinen to advance the development of new therapies for treating NASH. In addition to Sakari Kauppinen from Aalborg University, the NASH research team comprises the following members: professor Anders Näär, Department of Nutritional Sciences & Toxicology, University of California, Berkeley, USA; professor Pier Paolo Pandolfi, Cancer Center and Cancer Research Institute at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA; and associate professor Ryan Temel, Saha Cardiovascular Research Center, Department of Physiology, University of Kentucky, Lexington, USA.
Fatty liver disease affects 25% of the global population
Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease worldwide, with a global prevalence of 25%, and has been shown to be strongly associated with obesity and the metabolic syndrome. NASH is the progressive subtype of NAFLD, affecting more than 6% of adults in the United States.
The pathogenesis of NAFLD and NASH is complex and multifactorial, involving several mechanisms, including genetic, environmental and metabolic factors. NAFLD is caused by the accumulation of excessive fat in the liver cells, whereas NASH is characterized by the presence of ballooned hepatocytes, inflammation and liver fibrosis in addition to fat build-up in the liver.
“There is a strong link between obesity, metabolic syndrome and the development of NAFLD and NASH. As people’s waistlines continue to expand, we also see a steady increase in the prevalence of both NAFLD and NASH. This is a major public health problem, since NASH is rapidly becoming the leading cause of end-stage liver disease and liver transplantation,” says Sakari Kauppinen.
Early diagnosis is challenging
Although 25% of the global population is affected by fatty liver disease and therefore at risk of developing NASH, diagnostic tests and effective therapies for NASH are still lacking.
Further, if symptoms occur, they are often so vague and unspecific that many patients with NASH are unaware of their liver disease. Thus, a liver biopsy is required for making a definitive diagnosis of NASH.
Extensive research efforts are in progress worldwide to discover new non-invasive biomarkers for early diagnosis and to find better treatment options for NASH. As a result, hundreds of clinical trials on NASH are underway.
“Intercept Pharmaceutical’s lead drug Ocaliva® (obeticholic acid) is furthest ahead in clinical development and is expected to be approved for treating NASH in 2020. However, despite its demonstrated antifibrotic efficacy in NASH patients, Ocaliva® has many side-effects, and that is a potential concern,” says Sakari Kauppinen.
Worm genetics leads to discovery of the first microRNA
Like many other researchers, Sakari Kauppinen has entered the NASH field, but in contrast to most other approaches, the Aalborg University researcher will target microRNAs (miRNAs) to tackle NASH.
His long journey to miRNAs in NASH began in December 1993, when Victor Ambros and Gary Ruvkun published two seminal papers on the roundworm C. elegans in Cell.
In the course of studying the genetics of larval development in C. elegans, Victor Ambros and Gary Ruvkun focused their work on a gene called lin-4, which controls developmental timing in the roundworm. To their surprise, Ambros and Ruvkun found that the lin-4 gene product is not a regulatory protein, but instead encoded a tiny RNA that controls gene expression by binding to partly complementary sites on the 3’ untranslated regions of target mRNAs, thereby inhibiting translation.
This work marked the discovery of the first miRNA, lin-4, and uncovered an entirely new type of regulatory mechanism mediated by a small non-coding RNA.
“This was a groundbreaking discovery and showed for the first time that a tiny non-coding RNA molecule can have a big impact on roundworm development by regulating gene expression at the RNA level,” explains Sakari Kauppinen.
The human genome encodes 2,500 microRNAs
For many years, lin-4 was considered to be a genetic oddity in the worm, until a second miRNA, named let-7, with a similar function was discovered in C. elegans. Shortly thereafter, let-7 was found to be conserved across many vertebrate species, which prompted three research laboratories to undertake a cloning effort of small RNAs from three organisms. This work described for the first time numerous miRNAs in the roundworm, fruit fly and human HeLa cells and was published in October 2001 in three back-to-back articles, in Science.
These discoveries led to intense research in identifying additional miRNAs, which were soon discovered to be abundant in animals and plants and shown to control many important biological processes. In 2002, only a year after the seminal miRNA discoveries were published in Science, the first report suggesting a role of miRNAs in cancer was published. George Calin and Carlo Croce of Ohio State University were investigating genetic aberrations in B-cell chronic lymphocytic leukaemia (CLL) and discovered that a frequently deleted region of chromosome 13, called 13q14, contains two miRNA genes, miR-15 and miR-16, that were deleted or downregulated in more than 60% of CLL patients.
“We know now that the human genome encodes at least 2,500 miRNAs and that they are involved in regulating most if not all biological processes. We also know that they downregulate gene expression by inhibiting protein synthesis or promoting the degradation of target mRNAs and that one miRNA can regulate many mRNA targets and thus modulate multiple pathways,” says Sakari Kauppinen.
Treatment of hepatitis C virus infection by targeting a liver microRNA
Sakari Kauppinen got interested in miRNAs in 2001. Due to their small size, miRNAs are difficult to detect, and thus, Kauppinen initiated a research project at Exiqon to develop novel locked nucleic acid (LNA)-based methods for improved miRNA detection and analysis in animals and plants. LNA is a high-affinity RNA analogue invented in 1998 by Jesper Wengel from the University of Southern Denmark. Due to the high binding affinity of LNA toward complementary RNA, it turned out to be ideally suited for efficient and specific detection of small RNAs such as miRNAs.
The utility of LNAs in miRNA research was subsequently demonstrated by a Danish-Dutch team of scientists, who published the first comprehensive miRNA expression atlas in developing zebrafish embryos using LNA-modified miRNA detection probes synthesized by Exiqon. This study was published in Science in 2005.
Several important miRNA discoveries and the encouraging results achieved with LNAs in zebrafish led Sakari Kauppinen and scientists at the University of Copenhagen and the biotech company Santaris Pharma (now Roche Innovation Center Copenhagen A/S) to embark on a new endeavour to develop an LNA-based drug platform targeting disease-associated miRNAs for therapeutics. This project was launched in January 2006, when Sakari Kauppinen and colleagues formed a miRNA research consortium with funding from Innovation Fund Denmark.
The consortium decided to focus on a specific miRNA (named miR-122) that was completely conserved from zebrafish to humans and highly abundant and expressed almost exclusively in the liver. These characteristics made miR-122 especially attractive for therapeutic targeting, since lessons learned from previous research in antisense oligonucleotide drugs had shown that they accumulated efficiently in the liver. Further, in a seminal study published in 2005, Peter Sarnow and his team at Stanford University reported that miR-122 is an important host factor for the hepatitis C virus (HCV) and, hence, a potential therapeutic target for treating HCV infection, a leading cause of liver disease, with millions of HCV patients worldwide. These findings also launched Sakari Kauppinen’s interest in liver diseases.
“We already knew that miR-122 plays an important role in cholesterol metabolism. Moreover, a report from Peter Sarnow showed that miR-122 is important for hepatitis C virus propagation in liver cells. Hence, we decided to focus on miR-122 and quickly discovered a promising lead candidate, which we named miravirsen, that effectively antagonizes miR-122 activity. Importantly, we also showed that it is able to inhibit hepatitis C viral RNA accumulation in a cell model,” explains Sakari Kauppinen.
Subsequently, the miravirsen discovery team carried out several studies in rodents and non-human primates, demonstrating the efficacy, tolerability and safety of miravirsen in pre-clinical animal models (see the results here and here).
“Miravirsen was the very first miRNA-targeted drug that was advanced to studies in non-human primates and humans, and thus our work received a lot of interest from academia and the biopharmaceutical industry,” says Sakari Kauppinen.
Yet another important milestone in developing the first miRNA-targeted drug was achieved in 2013, when Santaris Pharma announced the successful completion of a Phase 2a study with miravirsen in HCV-infected patients. This study showed that treating patients with chronic HCV infection with miravirsen is safe and well tolerated and provides long-lasting antiviral activity without evidence of viral resistance. The study results were published in The New England Journal of Medicine.
Discovery of metabolic microRNAs in mice
The idea of miRNAs as therapeutic targets to treat NASH originates from long-term collaborative research efforts of Aalborg University, the University of California, Berkeley, Harvard Medical School and the University of Kentucky. Using genome-wide association studies and genetic studies in mice, the researchers discovered two metabolic miRNAs that function as key regulators of cholesterol, lipid and metabolic homeostasis.
Further, pharmacological inhibition of miRNA activity in high-fat-diet-induced obese mice was well tolerated and resulted in decreased liver steatosis, inflammation and fibrosis without affecting food intake.
“Our findings suggest that the two metabolic miRNAs could represent potential therapeutic targets for treating NASH. Moreover, we found that the levels of these miRNAs were upregulated in liver biopsies from NASH patients,” says Sakari Kauppinen.
These miRNA discoveries formed the basis for a joint grant application to the Novo Nordisk Foundation Challenge Programme to advance the discovery of new miRNA-targeted therapies for treating NASH.
Computer-aided design of microRNA inhibitors
During the 6-year project, the NASH research team will synthesize libraries comprising different miRNA inhibitor molecules, antimiRs, targeting the identified metabolic miRNAs.
The antimiR compound libraries will be designed using software developed at the Center for RNA Medicine of Aalborg University. The libraries will be screened in cell-based assays to identify the most potent compounds for pharmacological inhibition of the miRNAs. The efficacy and tolerability of the most promising lead compounds will subsequently be assessed in mouse models.
“Our goal is to validate these two miRNAs as targets for therapeutic intervention in NASH and to identify potent antimiR compounds that can effectively inhibit miRNA activity in cells and can therefore be advanced to preclinical studies in animal models of NASH,” says Sakari Kauppinen.
MicroRNA precision medicine
Another important objective of the project is to understand how metabolic homeostasis is controlled by the two miRNAs and to determine their functional contributions to the pathogenesis of NASH.
Sakari Kauppinen says that the NASH project was successfully kicked off in 2019 and that the team has already made good progress in identifying several promising antimiR compounds for validation in cell culture assays and animal models.
He also explains that binding affinity and specificity are important criteria when selecting the best lead compounds, which means that the antimiR compounds should specifically antagonize the miRNA without targeting other RNA molecules, leading to potential off-target effects.
It is thus of key importance to avoid off-target effects, which may lead to unintended side-effects. Off-target effects can be predicted by computer simulation when designing antimiR compounds, but should also be assessed experimentally in the laboratory.
“Our goal is to assess the efficacy of the lead antimiR compounds to treat NASH in relevant mouse and non-human primate models. In addition to being specific against the intended miRNA target, the compounds also have to be safe and well tolerated without any side-effects,” explains Sakari Kauppinen.
Studies in animal models
The NASH research team will also use mouse genetics to generate a variety of mouse models for studying the role of the two miRNAs. First, the researchers will generate knockout mice, in which the two miRNA genes are ablated, to gain insight into the in vivo roles of the metabolic miRNAs.
In addition, the NASH team will generate transgenic mice for the same miRNA genes to determine the phenotypic effects of their overexpression. Do such transgenic mice show accumulation of fat in the liver? Do they become obese? Does miRNA overexpression lead to inflammation or liver fibrosis?
The project will also deploy different diets in combination with the genetic loss-of-function and gain-of-function mouse models to explore the biological functions of the two miRNAs in regulating metabolic homeostasis and to investigate their role in promoting the development of NASH.
“We need to understand the biological role of the two metabolic miRNAs in normal physiology as well as in NASH. Thus, in addition to screening for potent inhibitors for pharmacological inhibition of miRNA activity, we need genetic mouse models to study miRNA function,” says Sakari Kauppinen.
New biomarkers with diagnostic potential
An independent goal of the research project is to evaluate miRNAs as potential diagnostic biomarkers for NASH.
The team has already established access to a large collection of liver biopsies and blood samples from NASH patients. The blood samples are especially interesting, since the expression levels and composition of circulating miRNAs have been shown to correlate with a variety of pathological conditions and hence could be a valuable resource for identifying non-invasive biomarkers for early diagnosis of NASH without the need for a liver biopsy.
“Circulating miRNAs have shown great promise as biomarkers in disease diagnostics and prognostics, and thus I hope that we will also be able to use them as non-invasive biomarkers in NASH,” says Sakari Kauppinen.
From bench to bedside
Sakari Kauppinen anticipates that the NASH research consortium will have identified and characterized at least one antimiR lead compound with demonstrated efficacy animal models of NASH without adverse effects by the end of the project period. Moreover, the team expects to identify circulating miRNAs that can be used as non-invasive biomarkers to diagnose NASH.
If a miRNA-targeted drug has advanced to clinical development for NASH when the project is completed after 5 years, then this endeavour will have been a great success.
“Our long-term goal is to translate basic research aiming to understand the role of miRNAs in regulating metabolic homeostasis into new therapeutic strategies for treating NASH. If this is successfully translated to the clinic, it could potentially have a great impact on public health worldwide,” says Sakari Kauppinen.