Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia

Abstract

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT^-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24 h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT^-/- newborn pup.

Keywords: GALT; IEM; POI; ataxia; classic galactosemia; galactose toxicity; galactose-1 phosphate; galactose-1 phosphate uridylyltransferase; inborn errors of metabolism; mRNA therapy; primary ovarian insufficiency; speech dyspraxia.

Figure 1

Disrupted Galactose Metabolism in Classic Galactosemia (A) Under normal conditions, exogenous and endogenously produced galactose are phosphorylated by galactokinase (GALK1) to form galactose-1 phosphate (gal-1P). In the presence of galactose-1 phosphate uridylyltransferase (GALT), gal-1P reacts with UDP-glucose to form glucose-1 phosphate and UDP-galactose. UDP-galactose can also be formed from UDP-glucose via the UDP galactose-4′-epimerase (GALE) reaction. (B) In Classic (GALT-deficiency) galactosemia, gal-1P accumulates and the production of UDP-galactose from the GALT reaction is blocked. Gal-1P is a competitive inhibitor of hUGP2 and therefore, UDP-glucose synthesis and its subsequent conversion to UDP-galactose (via the GALE reaction) are both diminished. Excess galactose accumulated in the GALT-deficient cells is catabolized to galactitol, which can be excreted in urine. Major organs affected in galactosemic patients are listed in the inset.

Figure 2

Expression and Activity of mRNA-Encoded mGalT in Patient Cells (A) Western blot analysis of mGalT protein in galactosemic patient fibroblasts. (B) mRNA-encoded mGalT enzyme activities in galactosemic patient fibroblasts. (C) Reconstitution of mGalT activity in human galactosemic patient fibroblasts. Data are presented as mean ± SD.

Figure 3

Expression and Activity of mGalT or hGALT in GalT-Deficient Mouse Liver 24 h after i.v. Injection (A) Western blot analysis of mRNA-encoded mouse and human GALT proteins in GalT-deficient mouse liver. (B) mRNA-encoded mouse GalT and human GALT enzyme activities in GalT-deficient mouse liver. Data are presented as mean ± SD.

Figure 4

Single i.v. Dose of mGalT mRNA Resulted in Long Half-Lived mGalT Protein and Activity in Liver and Reduced Hepatic and Tissue Gal-1P Accumulation (A) Western blot analysis of mRNA-encoded mouse GalT protein in GalT-deficient mouse liver over a course of 2 weeks after a single i.v. dose. (B) mRNA-encoded mouse GalT enzyme activity in GalT-deficient mouse liver over a single i.v. dose. (C) Galactose-1 phosphate levels in liver at different time points after single i.v. injection of mGalT mRNA. (D) Galactose-1 phosphate levels in RBCs at different time points after single i.v. injection of mGalT mRNA. Data are presented as mean ± SD. *p < 0.05 compared to correspondent control mRNA group from one-way-ANOVA followed by Sidak’s multiple comparisons test.

Figure 5

Repeated IV Dose of hGALT mRNA Reduced RBCs, Liver, Ovary, and Brain Gal-1P Accumulation and Decreased Plasma Galactose and Galactitol Accumulation (A) Galactose-1 phosphate levels in (i) RBCs, (ii) liver, (iii) ovary, and (iv) brain after repeated four doses of human GALT mRNA. (B) Plasma galactose levels after repeated doses of hGALT mRNA. Data are presented as mean ± SEM. *p < 0.05 compared to correspondent PBS group and #p < 0.05 compared to control mRNA group obtained from Dunn’s multiple comparisons test following a Kruskal-Wallis test (A, i and B). *p < 0.05 compared to PBS group from one-way-ANOVA followed by Sidak’s multiple comparisons test (A, ii, iii, and iv). # p < 0.05 compared to control mRNA group from one-way-ANOVA followed by Sidak’s multiple comparisons test (A, ii, iii, and iv).

Figure 6

Single i.p. Dose of Human GALT mRNA Reversed Neonatal Galactose Sensitivity and Promoted Growth (A) Percentage survival analysis of PBS, control GFP mRNA, and hGALT mRNA treated galactose intoxicated GalT-deficient pups. (B and C) Comparison of body weight among mutant pups treated with PBS, control GFP mRNA, and hGALT mRNA. The Mantel-Cox log rank test was used to compare the survival rates of pups in different groups (p < 0.0001).