Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of . Retinal organoids were generated using these cells, which retained gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and β, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.