P52

Molecular characterization of a non-coding mutation in GRN gene and investigation of CRISPR-dCas13 system for splicing modulation

A Velasco Bilbao(1) X Bujanda Cundin(1) O Arnold García(1) M García Puga(1) J Ondaro Ezkurra(1) L Blázquez García(1)

1:Biodonostia Institute

Mutations in granulin (GRN) gene are the second major genetic cause of frontotemporal lobar degeneration (FTD) and appear all throughout the gene. A non-coding mutation in the intron 7 of GRN, known as c.709-1G>A, has been reported to be specific of Basque population and some other rare cases in the world. However, the molecular mechanism by which this intronic variant causes FTD is unknown. In this work, we establish the effect of GRN c.709-1G>A mutation in silico, in vitro and in patient samples. We observe that the mutation leads to an aberrant splicing pattern which causes GRN mRNA degradation and progranulin haploinsufficiency. Next, we investigate whether the mutation is suitable for splicing modulation approaches using RNA-targeting CRISPR-dCas13 system. Similar to already approved splice-switching antisense oligonucleotides, this system binds to specific sequences in a target pre-mRNA, sterically blocks access of RNA-binding proteins and therefore modulates RNA-processing pattern. We investigate the use of CRISPR-dCas13 system to develop a personalized therapeutic approach which restores GRN reading-frame in order to generate a shorter but partially functional progranulin protein that can rescue FTD phenotype.