Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells

Abstract

Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5′-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.

DeepCLIP: predicting the effect of mutations on protein–RNA binding with deep learning

“…While some existing binding site prediction tools can work on sequences of any type, there is an unmet need for improved modeling of contextual dependencies other than structure that are important for correctly estimating the in vivo functionality of the binding sites. Extracted contextual information may form the basis for design of antisense oligonucleotide based therapies, which modulate RBP activity, such as splice-switching oligonucleotides (SSOs) ( 4–6 ). Thus, improving information on whether contexts act positively or negatively with regard to binding is an important area of research that will ultimately enable the development of novel therapeutic options in personalized medicine.…”

RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays

“…In vivo effect was also validated in a mouse model with ASO 7-26S which contains a more stable phosphorothioate backbone in structure. Similarly, the Andresen group also identified a splicing silencer in intron 20 which acts as the binding site of hnRNP A1, one splicing repressor [ 80 ]. Their most active ASO candidate, SSO1 which masks intron 20 +11 to +35 nucleotides was able to induce completely restoration of IKBKAP exon 20 inclusion and dramatic increase in IKAP protein levels in FD patient fibroblast cells at low nanomolar concentration.…”

Splicing isoform-specific functional genomic in cancer cells

“…Targeting an exclusively intronic sequence is less common but may be approached rationally by targeting putative intronic splicing regulatory sequence (Fig. 2d, f) [37][38][39]. Importantly, bioinformatic prediction of splicing regulatory sequences are used as guidelines for finding a robust SSO usually require testing several antisense sequences [40,41].…”