Description ​

Problem ​

Cirrhosis is a chronic progressive liver disease characterized by hepatic fibrosis and architectural distortion. Each year, cirrhosis and related liver diseases cause more than one million deaths worldwide, ranking as the 11th leading cause of death overall and the 3rd leading cause of death among individuals aged 45–64 years. Deaths attributable to cirrhosis and liver cancer account for 3.5% of global mortality¹, representing a substantial global health burden.

In China, there were 156,400 deaths from cirrhosis and 10.99 million new cases in 2021, accounting for 18.82% of the 58.40 million incident cases globally, with an upward trend. Notably, nonalcoholic fatty liver disease (NAFLD) has emerged as the leading cause of cirrhosis in China, making it a critical public health concern.² Patients frequently require repeated hospitalizations due to complications such as ascites, variceal bleeding, hepatic encephalopathy, and hepatocellular carcinoma, which significantly impair both quality of life and socioeconomic status.

Currently, the management of cirrhosis relies mainly on controlling underlying etiologies and managing complications. However, available therapies are limited in efficacy for advanced fibrosis, while liver transplantation remains restricted due to donor shortages. Therefore, the development of novel therapeutic approaches is urgently needed.

Shortcomings of Current Treatments ​

At present, clinical interventions for cirrhosis mainly focus on three strategies: pharmacological treatment, liver transplantation, and hepatoprotective therapy.

Pharmacological treatment mainly targets etiology, complications, and inflammation. However, this strategy cannot reverse the pathological structures of fibrosis and cirrhosis that have already formed; it can only delay disease progression, and due to impaired hepatic metabolic function, it carries potential risks related to drug use. Liver transplantation, which treats end-stage liver disease by removing the diseased liver and implanting a healthy one, is severely restricted by extreme shortages of donor organs. Moreover, the surgery is complex and risky, and patients must take lifelong immunosuppressive drugs, which lead to health problems associated with reduced immunity, as well as substantial economic burden.

In contrast, hepatoprotective therapies that directly target hepatocyte metabolism and functional restoration have demonstrated unique value—they delay the progression of cirrhosis through anti-inflammatory, antioxidant, detoxifying, and hepatocyte-repairing effects, aiming to correct metabolic disorders and repair damage at the cellular level. This has led us to select hepatoprotective therapy as the core direction for developing next-generation treatments for cirrhosis, in order to overcome the limitations of current therapeutic options.S-adenosyl-L-methionine (SAM) is a naturally occurring bioactive molecule present in human cells. As a hepatoprotective drug, it serves as an important adjunctive medication in clinical practice and plays critical roles in hepatocytes:

SAM is formed through the catalysis of methionine adenosyltransferase (MAT), which transfers the adenosyl group from triphosphate (pppi) to methionine. In patients with chronic liver disease, the MAT1A gene in hepatocytes is highly methylated, resulting in suppressed MAT1A expression⁴, significantly reduced SAM levels, and impaired oxidative stress defense and methylation reactions. This metabolic disorder aggravates liver injury and promotes the progression of fibrosis.

Clinical practice has confirmed that exogenous supplementation of SAM can, to a certain extent, delay the progression of liver disease; however, the existing supplementation therapy has obvious shortcomings:

Therefore, there is an urgent need for an innovative therapy that can achieve negative feedback regulation, thereby maintaining SAM concentration at normal physiological levels.

Inspiration ​

Introduction to RNA Therapy ​

As third-generation therapeutics, nucleic acid drugs differ from first-generation small-molecule drugs in that RNA-based therapies do not rely on the liver’s core metabolic enzyme systems and are degraded through more natural pathways, thereby imposing less metabolic burden on the liver. Compared with second-generation protein drugs, nucleic acid therapeutics possess a longer half-life, allowing a single administration to exert therapeutic effects for several weeks or even months, which greatly improves patient compliance.

Our goal ​

To develop an intelligent regulatory element, ReguSAMe, that can both sense SAM concentration signals and convert them into negative feedback effects to regulate SAM levels.

Our Solution ​

Tools ​

Sensor — Aptamer ​

Aptamers are short single-stranded nucleic acids that, due to their unique spatial conformation, can bind target molecules with high affinity and specificity. We obtained the SAM aptamer from the SAM VI riboswitch. Riboswitches are regulatory elements commonly found in the 5’-UTRs of bacterial mRNAs that can sense various small molecules and ions to regulate gene expression. They mainly consist of a ligand-binding aptamer domain (which is what we require) and an expression platform that regulates gene expression.⁷

To date, six types of SAM riboswitches have been discovered in nature. We chose the SAM VI riboswitch as the source of our aptamer because its structure is simple and well-defined, composed of only 55 nucleotides. Moreover, the SAM VI riboswitch binds SAM with a dissociation constant KD of only 2 μM⁸, indicating high affinity and providing an excellent foundation for further engineering.

Figure 4. SAM Aptamer

Signal Processing Module — HDV Ribozyme ​

Ribozymes are RNA molecules with catalytic activity. Here, we selected the ribozyme derived from the hepatitis delta virus (HDV). As a self-cleaving ribozyme, the HDV ribozyme catalyzes the cleavage of phosphodiester bonds within RNA, achieving structure-dependent self-cleavage.⁹ When the HDV ribozyme is inserted into the 3′UTR of an mRNA, the active ribozyme self-cleaves, removing the 3′ poly(A) tail that protects the mRNA, leading to mRNA degradation and loss of gene expression.

A common ribozyme engineering strategy is to insert an aptamer into one of the ribozyme’s stem-loop structures and to screen a large number of linker sequences connecting the aptamer and ribozyme in order to identify variants exhibiting optimal switching performance. The HDV ribozyme contains two modifiable stem-loop structures, and successful examples already exist in which HDV ribozymes have been engineered using theophylline and guanine aptamers. ¹⁰This is why we chose the HDV ribozyme.

Figure 5. HDV Ribozyme

Integration — Aptazyme ​

We replaced the P4-L4 region of the HDV ribozyme with the aptamer domain from the SAM VI riboswitch and optimized the linker sequence, obtaining a SAM-responsive aptazyme capable of sensing SAM and initiating self-cleavage.

Figure 6. Aptazyme

Effector — MAT1A ​

MAT1A is the gene encoding MAT I, an enzyme that catalyzes the activation of methionine to synthesize SAM. Increasing the expression level of the MAT1A gene can enhance the production rate of SAM, thereby replenishing intracellular SAM levels.

Figure 7. Role of MAT1A

Mechanism of Drug Action in Cirrhosis — ReguSAMe ​

We applied the SAM-responsive aptazyme to the 3′UTR region of MAT1A mRNA to regulate the translation level of MAT I, the enzyme responsible for SAM synthesis, thereby achieving the goal of modulating intracellular SAM concentration.

Figure 8. ReguSAMe

In hepatocytes with low SAM concentration, the riboswitch cannot bind SAM, and the ribozyme remains structurally incomplete and catalytically inactive. As a result, self-cleavage does not occur, and the mRNA retains its intact poly(A) tail, which protects it from degradation. Consequently, normal translation of the MAT I enzyme proceeds, catalyzing SAM synthesis.

When the intracellular SAM concentration reaches a normal level, the riboswitch binds to SAM and undergoes a conformational change, allowing the previously incomplete ribozyme to form a complete and catalytically active structure that initiates self-cleavage. After the mRNA’s poly(A) tail is cleaved, the mRNA is subsequently degraded within the cell, translation of the MAT I enzyme ceases, and the SAM concentration does not continue to rise.

LNP Delivery: ​

We plan to encapsulate the mRNA using lipid nanoparticles (LNPs) for liver-targeted drug delivery.

Currently, mRNA-3927, a liver-targeted therapeutic developed by Moderna for the treatment of propionic acidemia (PA), is in Phase I/II clinical trials. Its lipid composition (molar ratio of ionizable lipid : DSPC : cholesterol : PEG-lipid = 50 : 10 : 38.5 : 1.5) serves as a reference formulation. ¹¹Meanwhile, ongoing research in the field is focused on enhancing the hepatic targeting efficiency of LNPs—for example, by incorporating POPC as a helper lipid to improve hepatocyte selectivity.¹²