A4GALT-targeting siRNA lipid nanoparticles ameliorate Fabry disease phenotype: Greater efficacy in endothelial cells than in podocytes

In this study, we explore the therapeutic feasibility of globotriaosylceramide (Gb3) synthase (<i>A4GALT</i>)-specific siRNA-loaded polyhistidine (pHis)-incorporated lipid nanoparticles (HLNPs) for Fabry disease (FD). HLNPs were developed to deliver siRNAs targeting <i>A4GALT</i> using a microfluidic device, with pHis aiding in endosome escape. The therapy was tested on <i>GLA</i>-knockout human-induced pluripotent-stem-cell-derived endothelial cells (<i>GLA</i>-KO-hiPSC-ECs) and podocytes (<i>GLA</i>-KO-hiPSC-PCs). <i>GLA</i>-KO-hiPSCs-ECs or -PCs, upon differentiation, were treated with <i>A4GALT</i>-siRNA-HLNP. Successful intracellular uptake of <i>A4GALT</i>-siRNA-HLNP was confirmed through fluorescence and electron microscopy in both cell types. <i>A4GALT</i>-siRNA-HLNP treatment confirmed both cell types' stability at 5 μg/mL. Increased Gb3 deposition and zebra body formation were detected in both cell types, but <i>A4GALT</i>-siRNA-HLNP treatment attenuated these FD phenotypes, demonstrating reduced expression of <i>A4GALT</i> through western blot analysis. RNA sequencing analysis revealed that the expression of transcripts associated with FD was restored by <i>A4GALT</i>-siRNA-HLNP treatment in <i>GLA</i>-KO-hiPSCs-ECs, whereas in <i>GLA</i>-KO-hiPSCs-PCs, this effect was relatively less pronounced. Suppression of <i>A4GALT</i> via siRNA/HLNP treatment significantly rescued FD phenotypes especially in EC, presenting a novel therapeutic approach for FD.