Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP); this mutation leads to variable skipping of exon 20 and to a drastic reduction of ELP1 in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception; this loss leads to severe gait ataxia, spinal deformities, and respiratory insufficiency due to neuromuscular incoordination. There is currently no effective treatment for FD, and the disease is ultimately fatal. The development of a drug that targets the underlying molecular defect provides hope that the drastic peripheral neurodegeneration characteristic of FD can be halted. We demonstrate herein that the FD mouse TgFD9;IkbkapΔ20/flox recapitulates the proprioceptive impairment observed in individuals with FD, and we provide the in vivo evidence that postnatal correction, promoted by the small molecule kinetin, of the mutant ELP1 splicing can rescue neurological phenotypes in FD. Daily administration of kinetin starting at birth improves sensory-motor coordination and prevents the onset of spinal abnormalities by stopping the loss of proprioceptive neurons. These phenotypic improvements correlate with increased amounts of full-length ELP1 mRNA and protein in multiple tissues, including in the peripheral nervous system (PNS). Our results show that postnatal correction of the underlying ELP1 splicing defect can rescue devastating disease phenotypes and is therefore a viable therapeutic approach for persons with FD.

The defective gene DYS, which is responsible for familial dysautonomia (FD) and has been mapped to a 0.5-cM region on chromosome 9q31, has eluded identification. We identified and characterized the RNAs encoded by this region of chromosome 9 in cell lines derived from individuals homozygous for the major FD haplotype, and we observed that the RNA encoding the IkappaB kinase complex-associated protein (IKAP) lacks exon 20 and, as a result of a frameshift, encodes a truncated protein. Sequence analysis reveals a T-->C transition in the donor splice site of intron 20. In individuals bearing a minor FD haplotype, a missense mutation in exon 19 disrupts a consensus serine/threonine kinase phosphorylation site. This mutation results in defective phosphorylation of IKAP. These mutations were observed to be present in a random sample of Ashkenazi Jewish individuals, at approximately the predicted carrier frequency of FD. These findings demonstrate that mutations in the gene encoding IKAP are responsible for FD.

Familial dysautonomia (FD) is a currently untreatable, neurodegenerative disease caused by a splicing mutation (c.2204+6T>C) that causes skipping of exon 20 of the elongator complex protein 1 (ELP1) pre-mRNA. Here, we used adeno-associated virus serotype 9 (AAV9-U1-FD) to deliver an exon-specific U1 (ExSpeU1) small nuclear RNA, designed to cause inclusion of ELP1 exon 20 only in those cells expressing the target pre-mRNA, in a phenotypic mouse model of FD. Postnatal systemic and intracerebral ventricular treatment in these mice increased the inclusion of ELP1 exon 20. This also augmented the production of functional protein in several tissues including brain, dorsal root, and trigeminal ganglia. Crucially, the treatment rescued most of the FD mouse mortality before one month of age (89% vs 52%). There were notable improvements in ataxic gait as well as renal (serum creatinine) and cardiac (ejection fraction) functions. RNA-seq analyses of dorsal root ganglia from treated mice and human cells overexpressing FD-ExSpeU1 revealed only minimal global changes in gene expression and splicing. Overall then, our data prove that AAV9-U1-FD is highly specific and will likely be a safe and effective therapeutic strategy for this debilitating disease.

Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.