Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations

Abstract

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908-321C>G). AMOs were designed to target the newly created 5' splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence-specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild-type levels, we measured the amount of Ras-GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO-dependent decrease in Ras-GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been used successfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders.

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2

“…19 Later this technology was used successfully for other diseases like cystic fibrosis 20 or more recently for NF1. 21 Today, different pre-clinical and clinical splice-modulating therapies using AONs, like restoring open reading frames, influencing alternative splicing or inducing exon inclusion, have been successfully used for many different genetic disorders. 16,17 In this study, we report a NF2 patient with bilateral vestibular schwannoma who was found to bear a deep intronic mutation in the NF2 gene.…”

Recurrent Deep Intronic Mutations in the SLC12A3 Gene Responsible for Gitelman's Syndrome

“…Alleles with intronic mutations activating cryptic splicing sites are potentially correctable with antisense oligonucleotides to prevent the splicing machinery from recognizing the cryptic exon and promote normal splicing, as successfully shown in several genetic disorders (42)(43)(44). Similar antisense oligonucleotides may also apply to GS patients bearing deep intronic mutations and can be first tested on the leukocytes expressing NCC mRNA.…”

Comprehensive Genetic and Mutation Analysis of Familial Dementia with Lewy Bodies Linked to 2q35-q36

“…Once regarded as junk DNA, intronic and intergenic sequences are now perceived to contain crucial splicing machinery elements and other gene expression regulating motifs. Recently, several studies have shown that mutations disturbing these important elements can lead to disease [34][35][36][37]. Therefore, we expect that high throughput sequencing of the entire 3.3 Mb candidate region might reveal a more complex mutation associated with DLB in family DR246.…”