Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration

Abstract

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon–intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

A deep intronic variant inMMEcauses autosomal recessive Charcot-Marie-Tooth neuropathy through aberrant splicing

“…An FDA-approved "n-of-1" ASO, milasen, successfully blocked pathogenic pseudoexon inclusion in MFSD8 in a patient with neuronal ceroid lipofuscinosis type 7 94 . ASOs which block pathogenic pseudoexons, such as that created by MME c.1188+428A>G, have also been described in in vivo preclinical models 93,[95][96][97][98][99] . As such, individuals with the MME c.1188+428A>G variant may represent a form of CMT that is treatable through personalized ASO therapy and warrants further investigation.…”