Development and optimization of human glucocerebrosidase-encoding mRNA for Gaucher disease therapy.

Gaucher disease (GD) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in the GBA1 gene encoding the lysosomal hydrolase glucocerebrosidase (GCase). Mutations in GCase lead to glucosylceramide accumulation within macrophages. Current treatments, including enzyme replacement therapy (ERT) and substrate reduction therapy (SRT), alleviate symptoms but are limited by high cost, frequent dosing, and adverse effects, highlighting the need for novel strategies. To achieve higher expression levels and improved stability, we designed and optimized a series of hGBA1-mRNA by changing untranslated regions (UTRs), codon usage, and poly(A) tails, and evaluated their performance in vitro and in vivo. Optimized constructs achieved >6-fold higher GCase activity compared with the least efficient variants 24 h post-transfection in HEK293T and RAW264.7 cells, with an average half-life exceeding 54 h. The expressed enzyme localized to lysosomes and restored normal morphology and substrate accumulation in GBA1-knockout (KO) HEK293T cells. Following a single administration of hGBA1-mRNA encapsulated in lipid nanoparticles (LNPs) in wild-type FVB mice, GCase activity was detectable in the liver and spleen within 72 h. Our results demonstrate that optimized hGBA1-mRNA-LNPs can deliver functional human GCase in vivo, providing a promising and efficient mRNA-based therapeutic approach for GD.