mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

Glycogen storage diseases (GSDs) are a group of rare genetic disorders stemming from the inability to synthesize or break down glycogen due to enzyme defects. GSDIa, caused by glucose-6-phosphatase-alpha (G6Pase-α) deficiency, is characterized by severe hypoglycemia because G6Pase-α is crucial for converting glucose-6-phosphate (G6P) to free glucose, enabling glucose release from the liver. The absence of G6Pase-α results in life-threatening hypoglycemia during fasting, along with metabolic imbalances like lactic acidemia and hypertriglyceridemia, and organomegaly. Current standard-of-care, involving frequent feedings of cornstarch, is insufficient to prevent long-term complications such as hepatocellular adenomas (HCAs) and hepatocellular carcinomas (HCCs). Liver transplantation is the only curative option but carries high risks. Alternatives like stem cell infusion have transient effects, while viral vector-mediated gene therapies face challenges regarding sustained expression, genotoxicity, and neutralizing antibodies. Enzyme replacement therapy is also impractical due to G6Pase-α's hydrophobic nature and ER membrane localization. mRNA therapy offers advantages such as encoding any protein sequence, utilizing the cell's machinery for protein production and localization, and avoiding genomic DNA modification. However, efficient and safe delivery methods have been a hurdle, until recent advances in lipid nanoparticles (LNPs) for mRNA encapsulation. This study aims to evaluate the efficacy and safety of repeat-dosing mRNA therapy for GSDIa, addressing both hypoglycemia and the long-term risk of HCA/HCC.

Existing literature highlights the challenges in treating GSDIa. Current dietary management only partially addresses hypoglycemia and fails to prevent long-term liver complications. While some progress has been made with liver stem cell infusion and viral vector-mediated gene therapies, these approaches have limitations such as transient effects, genotoxicity concerns, and the risk of neutralizing antibodies. Enzyme replacement therapy is hindered by the unique properties of G6Pase-α, making it difficult to purify and deliver effectively. In contrast, mRNA therapy offers a promising alternative, providing a method to transiently restore G6Pase-α function without the risks associated with gene therapy.

The researchers optimized the mRNA sequence encoding human G6Pase-α using bioinformatics to identify highly conserved amino acid residues among mammalian orthologs. They evaluated several variants in HeLa cells, identifying the S298C variant as having improved expression and enzymatic activity. They confirmed the proper ER localization of this variant. The mRNA sequence was further optimized for codon usage to enhance protein expression and activity. The optimized mRNA (hG6PC S298C mRNA) was then encapsulated in LNPs for delivery. In vitro experiments were performed in Hep3B cells to assess expression and activity of the codon-optimized and non-codon optimized mRNA variants. In vivo studies utilized a liver-specific G6Pase-α knockout mouse model (L.G6pc-/-) that mirrors the human disease. Dose-ranging and duration-of-action studies assessed the effects of a single i.v. dose of hG6PC S298C mRNA-LNP on fasting blood glucose levels, liver morphology, liver weight, G6Pase-α protein and activity, and hepatic biomarkers. A repeat-dose study evaluated the long-term effects of multiple doses over several weeks. Safety assessments included measuring serum cytokine levels, liver enzymes (ALT), anti-drug antibodies (ADA), and monitoring for any signs of hypersensitivity or adverse effects. Finally, the researchers investigated the impact of hG6PC S298C mRNA on the prevention of HCA/HCC by feeding L.G6pc-/- mice a high-fat/high-sucrose diet to accelerate tumor development and then administering repeated doses of hG6PC S298C mRNA or eGFP mRNA. They analyzed the number and size of lesions, and levels of HCA/HCC-related biomarkers and genes.

The optimized hG6PC S298C mRNA showed significantly higher expression and enzymatic activity in both in vitro and in vivo settings compared to the wild-type mRNA. The mRNA had a relatively short half-life in the liver (20 hours), while the protein half-life was considerably longer (79 hours), indicating sustained therapeutic effect. In the L.G6pc-/- mouse model, a single dose of hG6PC S298C mRNA-LNP significantly improved fasting blood glucose levels in a dose-dependent manner, while also reducing liver weight, hepatic G6P, glycogen, and triglycerides, and serum triglycerides. The therapeutic effect lasted for at least 7 days after a single dose. Repeat dosing of hG6PC S298C mRNA-LNP (0.25 mg/kg) over 8 weeks maintained improved fasting blood glucose levels without adverse effects and with no significant increase in serum cytokine levels or anti-drug antibodies. Moreover, in a long-term HCC induction study, chronic treatment with hG6PC S298C mRNA significantly reduced the incidence and severity of HCA/HCC lesions in L.G6pc-/- mice, showing a beneficial effect on long-term disease pathology. Specifically, the number of tumor-bearing mice was drastically lower in the treatment group (~23% vs. ~58% in the control), as was the number of tumors per mouse and the overall tumor burden.

This study provides the first evidence that repeated administration of an mRNA-based therapy for GSDIa is both safe and effective in improving fasting tolerance and reducing hepatic lesions in a mouse model. The engineered, codon-optimized mRNA encoding the G6Pase-α S298C variant exhibited robust expression and activity, leading to normalization of metabolic parameters and a reduction in liver abnormalities. The transient nature of mRNA expression, combined with the observed lack of immune stimulation, suggests that this therapy has a favorable safety profile. This approach could potentially overcome limitations of current GSDIa treatments, including dietary management, stem cell therapy, and viral vector-mediated gene therapies, which may suffer from issues of efficacy, safety and durability. The ability of mRNA therapy to impact preexisting tumors warrants further investigation.

This research demonstrates the potential of mRNA therapy as a novel and effective treatment for GSDIa, addressing both acute hypoglycemia and long-term liver tumor development. The study's findings suggest that this approach could significantly improve the lives of patients with GSDIa. Future research should focus on larger animal models and ultimately clinical trials to confirm the findings and establish the optimal dosing regimen for human patients. The successful application of this technology could potentially extend its benefit to other hepatic metabolic enzyme deficiencies.

The study was conducted in a mouse model, and the results may not fully translate to humans. While the study demonstrated a reduction in HCA/HCC lesions, it did not definitively show a complete prevention or cure for these conditions, and the long-term effects of the treatment require further investigation. Further studies involving different dosing regimens and longer-term evaluations are needed to fully assess the efficacy and safety of this treatment.