Development of an oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia

**Background:** Familial dysautonomia (FD) is an autosomal-recessive neurodegenerative disease caused by a splicing mutation in the ELP1 gene, leading to tissue-specific skipping of exon 20 and reduced ELP1 protein. FD occurs almost exclusively in Ashkenazi Jewish individuals and is characterized by progressive gait ataxia, retinal degeneration, and autonomic dysfunction. There is no effective systemic therapy. Previous efforts identified kinetin and BPN15477 as splicing modulators, but they lacked sufficient potency and biodistribution. This study aimed to optimize a novel small molecule splicing modulator, PTC258, and evaluate its efficacy in a phenotypic FD mouse model.

**Methods:** PTC258 was synthesized and tested in FD fibroblasts for ELP1 splicing correction and protein production. In vivo efficacy was assessed in the TgFD9; Elp1^Δ20/flox^ mouse model, which recapitulates the tissue-specific mis-splicing and hallmark symptoms of FD. Treatment was started at birth by feeding dams and then weaned mice chow containing 0.002% (3 mg/kg/day) or 0.004% (6 mg/kg/day) PTC258. Gait was analyzed using the CatWalk XT system at 3 and 6 months. Retinal structure was assessed by spectral-domain optical coherence tomography (SD-OCT) measuring RNFL and GCIPL thickness. Retinal ganglion cell (RGC) survival was quantified by whole-mount staining with RBPMS. Dorsal root ganglia (DRG) volume and proprioceptive neuron counts were measured by immunohistochemistry for parvalbumin. ELP1 splicing and protein levels were quantified by RT-PCR, qPCR, and MSD immunoassay.

**Key Results:** PTC258 was approximately 30,000 times more potent than kinetin and 1,000 times more potent than BPN15477 in increasing ELP1 protein in FD fibroblasts (EC2X ELP1 protein = 10,000 nM for kinetin, 340 nM for BPN15477, and ~0.3 nM for PTC258). In TgFD9 mice, PTC258 increased full-length ELP1 transcript in a dose-dependent manner and led to at least a 5-fold increase in functional ELP1 protein in brain, trigeminal, liver, and quadricep. In the phenotypic FD mouse model, PTC258 treatment significantly improved gait ataxia at 6 months: stride length of front paws increased from 5.8 cm (vehicle FD) to 6.5 cm (0.002% PTC258) and 6.8 cm (0.004% PTC258); hind paw stride length increased from 5.5 cm to 6.2 cm and 6.5 cm, respectively. Base of support was completely rescued in the 0.002% group. SD-OCT showed that RNFL and GCIPL thickness were significantly reduced in vehicle FD mice at 3 and 6 months, and PTC258 treatment produced a dose-dependent improvement. For example, at 6 months in the central temporal region, RNFL thickness was 12.5 μm in controls, 8.0 μm in vehicle FD, 10.5 μm in 0.002% PTC258, and 11.2 μm in 0.004% PTC258. RGC counts at 6 months were significantly lower in vehicle FD mice in all quadrants; PTC258 treatment rescued RGC loss in a dose-responsive manner. DRG volume in FD mice was 60% of controls, and proprioceptive neuron number was reduced to 55% of controls; PTC258 treatment significantly increased both. ELP1 splicing correction was observed in brain, DRG, trigeminal, retina, and liver, with a 2-fold increase in functional ELP1 in the brain and a 1.5-fold increase in the DRG.

**Clinical Implications:** This study demonstrates that oral administration of PTC258, a highly potent and specific splicing modulator, can prevent the progressive neuronal degeneration characteristic of FD in a mouse model. The treatment rescued gait ataxia and retinal degeneration, two major debilitating aspects of the disease. These results provide critical preclinical efficacy data supporting the development of PTC258 as an oral therapy for FD. Given that 99.5% of FD patients share the same ELP1 splicing mutation, this approach could benefit all individuals with FD. Further safety and toxicity studies are needed before clinical translation.