PROJECT SUMMARY Given the recent FDA approval of targeted AAV gene therapy platforms and of small-molecule splicing modulators as treatments for genetic neurological disorders, our goal is to apply these powerful technologies to prevent the progressive optic neuropathy and blindness that develops in patients with the genetic recessive disease, Familial dysautonomia (FD). FD results from a splice site mutation in intron 20 of the gene ELP1 (formerly called IKBKAP). As a consequence of the mis-splicing, exon 20 is variably skipped, the mutant mRNA degraded, resulting in reduced levels of the encoded protein, Elp1.. Interestingly, the ability to splice the mutated pre-mRNA varies according to tissue type, with neurons least capable of splicing the mutated pre- mRNA. While the majority of the clinical deficits are due to the devastation of the sensory and autonomic nervous systems, as patients enter their teens, their macular retinal ganglion cells progressively die, manifesting as visual loss. Mouse that are null for Elp1 are embryonic lethal so the field has, until now, taken two distinct strategies to generate mouse models to investigate FD: (i) generation of conditional knock-out mice (CKO) using cell-type specific cre-driven promoters; and (ii) transgenic mice that contain the human FD ELP1 splicing mutation. The former approach has generated mouse models that recapitulate the FD optic neuropathy that results from the progressive death of retinal ganglion cells. These mice are an excellent pre- clinical model for testing the effectiveness of gene therapy for preventing the progressive demise of retinal ganglion cells (Aim 1A). However this model does not lend itself to testing the effectiveness of splicing enhancer compounds since it lacks the FD splicing mutation. The latter approach has culminated in the generation of transgenic mice that include copies of the human FD ELP1 mutated gene. These mice are asymptomatic unless they are crossed to a hypomorph or null background mouse, but these compound mice are typically too sick to investigate consistently. Here we will make a new “hybrid” line by crossing in the human FD ELP1 mutated gene into our retina-specific CKO line (Pax6-cre;Elp1flox/flox) to overcome these major challenges to the field. In so doing, we will generate a single mouse model that manifests the human FD optic neuropathy, in an otherwise healthy background, and contains the splice site mutation, which can be used to test a variety of therapeutic approaches (Aim1A, B). The overall aim of this proposal is to assess and compare two methods for restoring normal levels of the Elp1 protein in this new model mouse retinae using: (i) AAV2- mediated gene therapy (gene reintroduction) of the wild type Elp1 gene injected intravitreously, and (ii) a novel splicing enhancer compound that has been shown to promote the inclusion of exon 20 in the mutant FD gene in the retina, delivered orally through diet. Our goal is to test which method best...