Substrate reduction therapy based on siRNA for Fabry Disease

Resumen

Fabry Disease (FD) is a lysosomal storage disorder caused by mutations in GLA gene, resulting in an insufficient α-galactosidase A (α-Gal A) enzyme activity. Consequently, there is a progressive accumulation of glycosphingolipids, particularly globotriaosylceramide (Gb3) and its derivated lyso-Gb3 in vascular endothelial and smooth-muscle cells, specially in human heart and kidney. The available therapeutic options are limited to enzyme replacement therapy (Replagal® and Fabrazyme®), and the oral chaperone Migalastat®. These therapies increase α-Gal A activity, reducing the levels of Gb3 and lysoGb3. In this sense, substrate reduction therapy (SRT) is a new approach for limiting the formation of glycosphingolipids. SRT targeted to Gb3 synthase (encoded by the A4GALT gene) has been proposed as a new strategy for FD, as it leads to a reduction in Gb3 avoiding the depletion of other glycosphingolipids which do not accumulate in FD. Silencing therapies based on small interfering RNA (siRNA) are promising options as SRT tools. siRNA is a double-stranded RNA molecule, 21-25 nucleotides in length, able to cleave its complementary mRNA. The use of siRNAs requires the development of a vector able to protect it and to facilitate its interaction with the target cell, affording an adequate intracellular disposition of the nucleic acid. Lipid nanoparticles (LNP) are among the most widely non-viral nucleic acids carriers. LNP show several advantages, such as low or absence of in vivo toxicity, good long-term stability and the possibility to autoclave and sterilize. In fact, in 2018 FDA and EMA approved the first siRNA product, Onpattro®, and it was formulated in LNP. In this work, we have evaluated four different siRNA molecules targeted to the mRNA expressed by A4GALT gene. siRNAs were formulated in LNP as well as in a commercial transfection reagent. The silencing capacity was evaluated in IMFE-1 cells, a cellular line derived from primary endothelial cells obtained from FD patients.