A one and done therapeutic strategy to correct ELP1 splicing defect in familial dysautonomia - Abstract Familial dysautonomia (FD) is a neurodegenerative inherited disease caused by a splicing mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) that results in variable skipping of exon 20. This, in turn, leads to a drastic reduction of ELP1 protein in the central and peripheral nervous system. All FD patients possess at least one copy of the c.2204+6T>C mutation, and 99.5% of patients are homozygous for this mutation. Although individuals with FD suffer from multiple neurological symptoms, progressive blindness drastically reduces their quality of life and is a significant concern for patients and their families. Currently, there are no available treatments to stop the continuous neuronal loss and retinal degeneration characteristic of this devastating disorder. In the present study, we propose to develop a genome editing approach to correct the FD splicing mutation and restore the expression of functional ELP1 protein precisely and efficiently. Specifically, we will assess the therapeutic potential of restoring ELP1 expression in the retina with the goal of rescuing retinal degeneration in the FD mouse. In Aim 1, we propose to develop and customize base editors (BEs) to precisely correct the ELP1 mutation causing FD. We will first design split constructs for base editing capable of specific C-to-T editing in vivo via adeno-associated virus (AAV) delivery. In parallel, we will develop small Cas9 BEs that fit in a single AAV. Moreover, we will perform specificity assays to identify potential off-targets and apply additional engineering approaches in case we need to mitigate off-target editing in human cells. In Aim 2, we will assess the therapeutic effectiveness of our BE approach in rescuing retinal degeneration in vivo. We will identify the ideal dose of BEs delivered via AAVs in the retina of an asymptomatic humanized TgFD9 mouse for precise ELP1 on-target editing. Once we have selected the most optimal strategy, we will assess its therapeutic efficacy in rescuing retinal degeneration in a humanized FD mouse that recapitulates the same retinal ganglion cell loss observed in FD patients. In summary, this proposal will explore novel genome editing technologies as permanent treatments for FD, offering a solution to rescue retinal degeneration leading to blindness. More importantly, this proposal aligns with our long-term goals of leading a translational research program towards a durable single-dose therapeutic strategy to correct the ELP1 splicing defect in FD patients, and it is rationally designed to provide essential ‘proof of concept’ in vivo data necessary for the success of this program.
