Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia

Abstract

Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin's ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis.

Modulation of aberrant NF1 pre-mRNA splicing by kinetin treatment

“…[3][4][5][6][7][8] This modification has mainly been shown in splicing mutations giving a percentage of wildtype transcripts from the mutated allele, which is also described as having a 'leaky' effect. In a previous collaborative study in which kinetin corrected aberrant splicing for the most common splicing mutation in familial dysautonomia (FD), we also demonstrated that kinetin was able to restore normal splicing for one NF1 mutation (c.6724C4T, exon 36) in a minigene splicing model, 9 which encouraged us to extend our research to other mutations. In this study, we evaluated the effect of kinetin and of four other drugs (valproic acid (VPA), sodium butyrate (SB), (À)-epigallocatechin gallate (EGCG) and aclarubicin), which were previously found to modify splicing in other genetic disorders, in cell lines derived from patients harboring 19 different NF1 splicing mutations to evaluate their putative use as therapeutic agents for NF1.…”

“…Interestingly, cell lines from the patient with mutation c.6724C4T showed less evident exon 36 inclusion that the minigene model from our previous study. 9 Differences in the splicing behavior of minigenes and primary cell lines have been reported in literature, which highlights the importance of the surrounding genomic region in determining the splicing. 10,11 Moreover, the heterozygosity of cell lines derived from patients vs homozygosity in constructions should be taken into account when comparing data.…”

“…In two cases, fibroblast cultures from skin or neurofibromas were also obtained. Cell lines were grown as previously described 8,9 and were treated with the following concentrations: kinetin (K, 100 mM), VPA (50 mM), SB (5 mg/ml), EGCG (25 mg/ml), all dissolved in water, and aclarubicin (A, 10 nM) dissolved in DMSO (see details in the Results section). Nontreated cell lines or cells treated with DMSO were used as controls.…”

“…Experiments were performed in duplicate. The relative amount of NF1 full-length and aberrantly spliced transcripts was determined using a semiquantitative RT-PCR approach and an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) 9 (Supplementary Figure 3). Results are shown as percentages of aberrantly spliced transcripts.…”

A Targeted Deleterious Allele of the Splicing Factor SCNM1 in the Mouse

“…There is evidence that the clinical severity of these disorders is influenced by splicing efficiency. Therapeutic strategies that target the correction of splicing defects are under development for spinal muscular atrophy, familial dysautonomia, and cystic fibrosis (Nissim-Rafinia et al 2004;Hims et al 2007;Hua et al 2008). The molecular basis for individual variation in splicing efficiency is largely unknown, but identification of the responsible factors could facilitate the development of therapies.…”