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

Abstract

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Conflict of interest statement

J.C., M.C., E.G., E.D., J.Z., S.L., L.Y., A.F., A.-R.G., K.B., T.K., C.M., Z.Z., B.L., G.B., M.W., B.T., C.T., E.O., J.S., A.D., V.P., C.P., W.Z., L.C., M.H., E.M., L.R., V.N., M.Z., U.R., P.F.F., P.G.V.M., and P.H.G. are employees of and receive salary and stock options from Moderna Inc. M.Si., M.So., V.V., E.W., A.M., G.M., and F.R. declare no competing interests.

Figures

Fig. 1. In vitro characterization of modified mRNA encoding hG6Pase-α.

a Hypothetical model of hG6PC mRNA therapy. hG6PC mRNAs are delivered to liver via lipid nanoparticles. Once the mRNA is in the cell (hepatocytes) it is translated by the cellular machinery into a functional protein that is localized to the ER membrane (likely following a co-translational translocation model), resulting in an active G6Pase-α enzyme. b Protein consensus screening by ortholog residue analysis. Top: WebLogo representation of the abundance of each alternative amino acid used at indicated residue positions. Bottom: The degree of conservation of amino acids at each position was quantified as relative entropy (Kullback–Leibler divergence). c Relative hG6Pase-α protein expression (solid circle) and hG6Pase-α enzymatic activity (solid square) in HeLa cells treated with the top ten hG6PC mRNA variants generated using protein consensus analysis. Data were shown as percentage of wild-type (WT) group and presented as mean ± SD of n = 2 (for protein expression), 3 (for enzymatic activity, Q247R), or 4 (for enzymatic activity, all other groups) biologically independent samples. d Subcellular localization of WT hG6Pase-α and S298C variant in HeLa cells. Green: hG6Pase-α, Red: Calnexin, an ER marker (top); TOM20, mitochondrial marker (bottom). Scale bars are 10 µm. The ratio of colocalized signal over total signal was calculated by Mander’s colocalization coefficient analysis (bottom panel). Data were presented as mean ± SD of n = 2 biologically independent samples. Source data are provided as a Source Data File.

Fig. 2. Effect of codon optimization on expression and activity of hG6PC mRNAs.

a hG6Pase-α protein expression (left panel) and enzymatic activity (right panel) of wild-type hG6PC (WT), codon optimized wild-type hG6PC (WT_CO), hG6PC_S298C (S298C), and codon optimized hG6PC_S298C (S298C_CO) mRNAs evaluated in Hep3B cells. Control cells were treated with eGFP mRNA. Data were presented as mean ± SD of n = 3 biologically independent samples. b hG6Pase-α protein expression (left panel) and enzymatic activity (right panel) of WT and codon optimized hG6PC mRNAs as evaluated in male CD-1 mice. Control animals were treated with eGFP mRNA. Data were presented as mean ± SD of n = 4 mice. For statistical analysis, raw values were Log2 transformed and subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the non-codon optimized WT hG6PC mRNA. Statistically significant P values (p ≤ 0.05) are shown in the graphs. Source data are provided as a Source Data File.

Fig. 3. Hepatic hG6PC mRNA and hG6Pase-α protein and activity half-lives in wild-type mice.

Wild-type (CD-1) male mice were i.v. administered with 1.0 mg/kg of eGFP, hG6PC-wild type (WT), or codon-optimized hG6PC-S298C (hG6PC_S298C_CO) mRNA-LNP and sacrificed at 6, 24, 72, 168, 336 h (n = 4/group/sacrifice time point). a hG6PC mRNA levels (hG6PC-WT and S298C mRNAs). b Hepatic protein levels in mice treated with eGFP mRNA, mRNA encoding hG6Pase-α WT, or codon-optimized mRNA encoding hG6PC-S298C protein variant. c Hepatic enzymatic activity levels in mice treated with eGFP mRNA, mRNA encoding hG6Pase-α WT, or codon-optimized mRNA encoding hG6PC-S298C protein variant. Data were presented as mean ± SD (n = 3–4). Source data are provided as a Source Data File.

Fig. 4. Single i.v. dose of hG6PC S298C mRNA-LNP restores euglycemia, as well as serum and hepatic biomarkers in L.G6pc−/− mice.

a Blood glucose levels following administration of hG6PC S298C mRNA-LNP in L.G6pc−/− mice. WT, wild-type mice. (WT treated with PBS, n = 8 per group; L.G6pc−/− treated with eGFP, n = 6, 5, 5, and 5 per group for fasting duration of 0, 2.5, 6, and 24 h, respectively; L.G6pc−/− treated with hG6PC S298C at 0.2 mg/kg, n = 7 per group for all time points; L.G6pc−/− treated with hG6PC S298C at 0.5 mg/kg, n = 7, 6, 6, and 6 per group for fasting duration of 0, 2.5, 6, and 24 h, respectively; L.G6pc−/− treated with hG6PC S298C at 1.0 mg/kg, n = 7 per group for all time points). Data were presented as mean ± SD. b Liver morphology (left panel) and liver weight (right panel) following administration of hG6PC S298C mRNA in L.G6pc−/− mice. Representative liver images are shown from n = 8, 5, 7, 6, and 6 mice per group from WT treated with PBS, L.G6pc−/− treated with eGFP, and L.G6pc−/− treated with hG6PC S298C mRNA at 0.2, 0.5, or 1.0 mg/kg, respectively. c hG6Pase-α S298C protein expression and enzymatic activity in livers of L.G6pc−/− mice. d Hepatic biomarker analysis following administration of hG6PC S298C mRNA-LNP in L.G6pc−/− mice. Liver G6P (left panel), liver glycogen (middle panel), liver triglycerides (right panel). e Serum triglycerides following administration of hG6PC S298C mRNA-LNP in L.G6pc−/− mice. hG6PC S298C mRNA-LNP dose range: 0.2, 0.5, and 1.0 mg/kg. For b–e, quantitative data were presented as mean ± SD (n = 8, 5, 7, 6, and 6 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, and L.G6pc−/− treated hG6PC S298C mRNA at 0.2, 0.5, or 1.0 mg/kg, respectively). For statistical analysis, raw values were Log2 transformed and subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the eGFP mRNA treated group. Statistically significant P values (p ≤ 0.05) are shown in the graphs. Source data are provided as a Source Data File.

Fig. 5. Repeat i.v. dose of hG6PC mRNA-LNP results in safe and effective restoration of euglycemia in L.G6pc−/− mice.

a Single-dose duration of action of hG6PC S298C mRNA-LNP (0.5 or 1.0 mg/kg) administered i.v. in L.G6pc−/− mice. Blood glucose levels were measured at fed (0 h) or fasting conditions (2.5- or 6-h post-fasting). Data were presented as mean ± SD (n = 8, 9, 10, 10, and 10 mice per group for wild-type (WT) treated with PBS, L.G6pc−/− treated with eGFP, hG6PC S298C mRNA at 0.5 or 1.0 mg/kg, respectively). For statistical analysis, two-sample t-test (two-sided) was performed and corrected for multiple testing by using a Bonferroni adjusted level of 0.005. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, comparing hG6PC S298C mRNA 1.0 mg/kg with eGFP (p values are 0.0017 [day 0, 2.5 h], 0.0016 [day 0, 6 h], 0.0006 [day 2, 2.5 h], 0.0005 [day 2, 6 h], 0.001 [day 4, 2.5 h], 0.001 [day 4, 6 h], and 0.048 [day 7, 6 h], respectively). †P ≤ 0.05, ††P ≤ 0.01, †††P ≤ 0.001, ††††P ≤ 0.0001, comparing hG6PC S298C mRNA 0.5 mg/kg with eGFP (p values are 0.033 [day 0, 2.5 h], 0.001 [day 0, 6 h], 0.0004 [day 2, 2.5 h], 0.00003 [day 2, 6 h], 0.002 [day 4, 2.5 h], 0.001 [day 4, 6 h], and 0.009 [day 7, 6 h], respectively) b Blood glucose levels following repeat (five doses) i.v. administrations of hG6PC S298C mRNA-LNP (0.25 mg/kg) in L.G6pc−/− mice. Arrows indicate dose administration. Blood glucose levels were measured at 2.5-h post-fasting. Data were presented as mean ± SD (n = 8, 7, and 9 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, and L.G6pc−/− treated with hG6PC S298C mRNA, respectively). For statistical analysis, two-sample t-test (two-sided) was performed and corrected for multiple testing by using a Bonferroni adjusted level of 0.005. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ***P ≤ 0.0001 comparing hG6PC S298C mRNA with eGFP (p values are 0.005 [day 11], 4 × 10−6 [day 25], 0.0007 [day 28], 0.0192 [day38], 1.3 × 10−5 [day 39], 0.001 [day 42], 2 × 10−6 [day 52], and 5 × 10−5 [day 53], respectively). c Serum proinflammatory cytokines (from left to right): IFNɣ, IL-1β, TNFα, and IL6 from the dose-ranging study. d serum ALT (mU/mL) levels from the dose-ranging study. For c and d, data were presented as mean ± SD (n = 6, 5, 8, 7, and 6 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, and L.G6pc−/− treated with hG6PC S298C mRNA at 0.2, 0.5, or 1.0 mg/kg, respectively). e Serum proinflammatory cytokines (from left to right): IFNɣ, IL-1β, TNFα, and IL6 from repeat-dose study. Data were presented as mean ± SD (n = 10, 10, and 7 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, or hG6PC S298C mRNA). f Antidrug antibody assay measuring anti-G6Pase-α antibodies in sera of mice treated with five doses of hG6PC S298C mRNA-LNP (0.5 mg/kg). Data were presented as mean ± SD (n = 9, 7, 7, 6 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, L.G6pc−/− treated with hG6PC S298C mRNA, and positive sera, respectively). g Body weight of L.G6pc−/− mice prior to each repeat i.v. dose treatment of hG6PC mRNA -LNP (0.25 mg/kg) for repeat dose study. Data were presented as mean ± SD (n = 8, 7, and 9 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, and L.G6pc−/− treated with hG6PC S298C mRNA, respectively). For statistical analysis of c–f, raw values were Log2 transformed and subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the eGFP mRNA treated group. P values are shown in the graphs (c–f). Source data are provided as a Source Data File.

Fig. 6. Effect of hG6PC S298C mRNA-LNP on prevention of hepatic adenomas in L.G6pc−/− mice.

a Number of mice with tumors (left), number of tumors per mouse (middle), and tumor burden/area (right). Data were presented as mean ± s.e.m (n = 21, 26, and 34 mice per group for wild-type (WT) treated with PBS, L.G6pc−/− treated with eGFP, and hG6PC S298C mRNA, respectively). b Liver morphology (tumor-circled in yellow) (top panels) and liver histology (bottom panels) of WT and L.G6pc−/− mice treated with either eGFP mRNA or hG6PC S298C mRNAs. c HCA/HCC biomarkers (protein expression). Results are expressed as mean ± SD (n = 15, 27, and 35 mice per group for WT treated with PBS, L.G6pc−/− treated with eGFP, and L.G6pc−/− treated with hG6PC S298C mRNA, respectively). For statistical analysis, raw values were subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the eGFP mRNA treated group. Statistically significant P values (p ≤ 0.05) are shown in the graphs. Source data are provided as a Source Data File.