Dissection of the function of Lebercilin and its dys-function in Lebers Congenital Amaurosis

01.10.2019 bis 30.09.2020

Leber Congenital Amaurosis (LCA) is a severe and early onset blinding disease. It affects children in the first year of life and is the most common cause of blindness at this age. We have previously shown that mutations in Lebercilin cause LCA due to an impaired transport along cilia in photoreceptors which leads to rapid degeneration and thereby blindness. Yet, the molecular function of Lebercilin and especially why IFT in photoreceptors is impaired is unclear. This could be due to a defect in photoreceptors, yet it could also be a consequence of Lebercilin dysfunction in the RPE. To gain insights into the molecular function of Lebercilin, CRISP/Cas9 was used to generate hTERT-RP1 knock-out cell lines and to introduce endogenous FLAG tags in HEK293T cells. Further, BioID2 was applied to characterize transient interactions of Lebercilin and its complex components. The knock-out of Lebercilin in hTERT-RP1 resulted in a clear cilia disassembly phenotype. After initiation, cilia disassembly was normal in control cells while in knock-out cells, it was strongly impaired. This might be explained by the interplay between Lebercilin and novel interactors that are central components of the ciliary disassembly machinery. This project aims at validating these findings and identifying the molecular mechanisms to understand the role of Lebercilin as well as cilia disassembly in the retinal phenotype in LCA. To achieve this, we will employ established technologies, namely CRISPR/Cas9-based genomic editing to knock-out components of the Lebercilin protein network as well as imaging and protein complex analysis. Dissecting the molecular mechanisms is essential for understanding LCA and for development of treatment. If for example gene therapy targets photoreceptors but the initial phenotype develops in cells of the retinal pigment epithelium, the therapy will not be effective.