Novel MicroRNAs Targeting Hepatic Stellate Cells in Treatment of Fibrosis in Progressive Liver Diseases

Executive summary

Most progressive liver diseases cause morbidity and mortality through the activation of hepatic stellate cells (HSCs). HSCs promote fibrosis, the formation of fibrous tissue which leads to extensive liver scarring (cirrhosis) and end-stage liver disease. Reversing or preventing HSC activation could be a way to prevent fibrotic disease progression in many liver diseases, such as non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD).

Currently, there is no drug that directly targets HSCs. Knowing this, the inventors screened for and discovered microRNAs (miRNAs) that reverse the activation of HSCs. MicroRNAs are small, single-stranded, non-coding RNA molecules that play an important role in gene expression. The inventors identified two lead miRNA molecules—miR-15a and miR-412—revert activated mouse and human HSCs back to quiescence in ex vivo mouse and human models of liver disease. This reversion prevents these HSCs from inducing signs of liver disease, such as hepatocyte inflammation and steatosis.

Unmet need

Currently, there are no treatment options for many progressive liver diseases and none that target HSCs. Liver transplant is oftentimes the only treatment option for advanced stages of liver disease and liver failure. Not every patient is eligible for such a procedure, further emphasizing the need for treatment options for progressive liver diseases. Additionally, risk factors for NASH include obesity, diabetes, and hyperlipidemia, and most patients struggle to avoid these risk factors which leads to a critical need for effective treatment.

Value proposition

The use of miRNAs or their analogues is a novel therapeutic approach for liver diseases. Therefore, this discovery could serve as the foundation for developing miRNAs as a novel therapeutic option that could regulate HSCs and decrease liver fibrosis in many different liver diseases.

Non-alcoholic fatty liver disease (NAFLD) is the most common, and it effects 80 million Americans. NAFLD, which can lead to NASH, is also now the most common hepatic disorder in industrialized nations. Furthermore, in the next 10 years, it is likely to become the most common cause of end-stage liver disease, hepatic transplant, and hepatocellular carcinoma. With a large patient base and unmet need, this technology has a unique opportunity to become the first and one-of-its-kind treatment option for this and other fibrotic diseases of the liver.

Rationale of disease

Background

Progressive liver diseases, including NAFLD, PSC, and ALD, could lead to cirrhosis of the liver. Cirrhosis is a late stage of scarring of the liver, causing symptoms of jaundice, altered mental status, fluid retention, and lethal gastrointestinal bleeding. Treatment options are limited, and those that exist focus on treating the underlying cause (excessive alcohol consumption, hepatitis, for example) to prevent further damage to the liver.

Unmet needs

Treatment options are limited for patients with progressive liver disease. Treatment is focused on prevention, so there are no treatments that exist to reverse liver damage.

Technology

Background

This technology focuses on the activity of HSCs in the liver. When these cells are activated in response to liver injury or chronic inflammation, they turn into myofibroblasts to promote fibrosis. Left untreated, fibrosis leads to cirrhosis, and eventually, end-stage liver disease. The inventors created a novel miRNA treatment for progressive liver diseases that prevent or even reverse the activation of HSCs. miRNAs are small, non-coding RNA molecules, but they are known to have the ability influence an entire cellular program by targeting multiple genes. After extensive screening, the inventors found that miR-15a and miR-412 effectively revert activated HSCs to quiescence in human and mouse models. This therapy will be used in conjunction with existing preventative therapies to reduce steatosis, hepatitis, and fibrosis, which are all hallmarks of progressive liver disease.

Advantages and progress

Various factors contribute to liver inflammation, including viral and bacterial infections, metabolic disorders, toxins, and dietary factors. Growing evidence indicates that miRNAs have a fine-tuning role in liver inflammation by targeting various signaling molecules. A plethora of miRNAs have been implicated in inflammatory responses. Thus, the identification of these miRNAs is a novel approach to treat liver diseases.

Proof of concept

HSC morphologic differences determined by the activation status

Quiescent HSCs in low and high magnification. The high magnification view clearly shows several lipid droplets that fluoresce green with BODIPY stain.

Activated HSCs in low and high magnification. Activated cells are much larger, lack lipid droplets, and do not stain with BODIPY.

The effect of miR-15a on hepatocytes

miR-15a delivered to hepatocyte caused no significant expressional changes in hepatocyte markers, analyzed by PCR. Aat, alpha1-antitrypsin; Alb, albumin; Ttr, transthyretin; Tdo, tryptophan 2,3-dioxygenase; Ggt, gamma-glutamyltransferase; G6p, glucose-6-phosphatase; Cyp1a1, cytochrome P450 family 1 subfamily a polypeptide 1; Cyp3a11, cytochrome P450 family 3 subfamily a polypeptide 11; Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

miR-15a delivered to hepatocyte caused no significant expressional changes in albumin analyzed by ELISA. Data are mean ± SEM., unpaired two-tailed t-test, n=6.

miR-15a caused expression levels of most pro-inflammatory cytokines checked to either stay unchanged or downtrend (ns, not significant. Data are mean ± SEM., unpaired two-tailed t-test, *P < 0.05, n=3).

miR-15a and miR-412 were administered as mimics or expressed by vector systems in activated HSCs. Administration of miRNA by either method to activated HSCs caused 10-100-fold decrease in size, typical of quiescence. HSCs can be permanently reprogrammed into a quiescent state through piggyBac vectors that integrate into the genome.

Cell therapy with reprogrammed, quiescent-like HSCs improved liver steatosis, inflammation, and fibrosis in both in vitro and in vivo settings. Forced expression of either miRNA downregulated two HSC activation markers including alpha smooth muscle actin (Acta2), alpha-1 type I collagen (Col1a1), and others.