Novel mRNA therapy restores GALT protein and enzyme activity in a zebrafish model of classic galactosemia

Abstract

Messenger RNA (mRNA) has emerged as a novel therapeutic approach for inborn errors of metabolism. Classic galactosemia (CG) is an inborn error of galactose metabolism caused by a severe deficiency of galactose-1-phosphate:uridylyltransferase (GALT) activity leading to neonatal illness and chronic impairments affecting the brain and female gonads. In this proof of concept study, we used our zebrafish model for CG to evaluate the potential of human GALT mRNA (hGALT mRNA) packaged in two different lipid nanoparticles to restore GALT expression and activity at early stages of development. Both one cell-stage and intravenous single-dose injections resulted in hGALT protein expression and enzyme activity in the CG zebrafish (galt knockout) at 5 days post fertilization (dpf). Moreover, the levels of galactose-1-phosphate (Gal-1-P) and galactonate, metabolites that accumulate because of the deficiency, showed a decreasing trend. LNP-packaged mRNA was effectively translated and processed in the CG zebrafish without signs of toxicity. This study shows that mRNA therapy restores GALT protein and enzyme activity in the CG zebrafish model, and that the zebrafish is a suitable system to test this approach. Further studies are warranted to assess whether repeated injections safely mitigate the chronic impairments of this disease.

Keywords: GALT; classic galactosemia; lipid nanoparticles; mRNA; therapy; zebrafish.

FIGURE 1

Study design. Naked, LNP1 and LNP2‐packaged hGALT mRNAs provided by Moderna Inc. were injected in CG zebrafish (galt KO) zebrafish at one‐cell stage (0–1 h post fertilization, hpf) and intravenous (48–56 hpf). GALT activity, protein expression (merely one‐cell stage injections) and Gal‐1‐P and galactonate (merely intravenous injections) were assessed at 5 days post fertilization (dpf). LNP, lipid nanoparticle; mRNA, messenger RNA; hpf, hours post fertilization; dpf, days post fertilization; LNP1, LNP1‐packaged mRNA; LNP2, LNP2‐packaged mRNA; Naked, naked mRNA; Gal‐1‐P, galactose‐1‐phosphate. Figure is created with BioRender.com

FIGURE 2

hGALT expression and activity in 5 dpf CG zebrafish (galt KO) after single dose hGALT mRNA injection (100 ng/μl) at the one‐cell stage. A: Western blot analysis: Non‐injected WT and CG zebrafish (galt KO) controls present no band, which indicates that the antibody is specific for hGALT. Samples were loaded in duplicate (n = 150 per sample). B: GALT activity (nmol/mg protein/h), results are presented as mean ± SEM. All samples (n = 3 per experimental group, on average 30 zebrafish/sample), were measured in duplicate. MW, molecular weight ladder; WT, wildtype; NIC, non‐injected CG zebrafish (galt KO) control; NKD, naked mRNA injected CG zebrafish (galt KO); LNP1, LNP1‐packaged hGALT mRNA injected CG zebrafish (galt KO); LNP2, LNP2‐packaged hGALT mRNA injected CG zebrafish (galt KO)

FIGURE 3

hGALT activity in 5 dpf CG zebrafish (galt KO) after single dose intravenous hGALT mRNA injection (100 ng/μl). Results are presented as mean ± SEM. All samples (n = 6 for wildtype zebrafish and n = 4 for NIC and LNP2 injected zebrafish, ~90 zebrafish/sample), were measured in duplicate. WT, wild‐type; NIC, non‐injected CG zebrafish (galt KO) control; LNP2, LNP2‐packaged hGALT mRNA injected CG zebrafish (galt KO)

FIGURE 4

Gal‐1‐P and Galactonate in 5 dpf CG zebrafish (galt KO) after single dose intravenous hGALT mRNA injection (100 ng/μl). A. Gal‐1‐P (peak area) B. Galactonate (peak area)Results are presented as mean ± SEM. C, D, E. Gal‐1‐P levels and corresponding logarithm of GALT activity levels (nmol/mg protein/h) Sample size per condition (unexposed or exposed to galactose) was n = 6 for WT zebrafish and n = 4 for NIC and LNP2 injected zebrafish, ~90 zebrafish/sample see Table S1). WT, wild‐type; NIC, non‐injected CG zebrafish (galt KO) control; LNP2, LNP2‐packaged hGALT mRNA injected CG zebrafish (galt KO)

FIGURE 5

Galactose (100 mM) exposure: survival rates in 5 dpf CG zebrafish (galt KO) following single dose intravenous hGALT injection (100 ng/μl). Total sample size at the start of galactose exposure can be found in Table S2, results are presented as survival rate ± 95% CI. WT, wildtype; NIC, non‐injected CG zebrafish (galt KO) control; LNP2, LNP2‐packaged hGALT mRNA injected CG zebrafish (galt KO)