A CEP290 C-terminal protein fragment rescues retinal degeneration in a mouse model of Leber Congenital Amaurosis

Abstract

Purpose : Leber Congenital Amaurosis (LCA) is an inherited retinal degenerative disorder affecting 1 in every 80,000 live births. Mutations in CEP290 gene account for approximately 22% of total LCA cases. Gene replacement therapy for CEP290-LCA was hindered by the large size of the wild-type (WT) CEP290 coding sequence that exceeds the packaging limit of an adeno-associated viral (AAV) vector. To circumvent this problem, we tested whether a part of the CEP290 coding sequence can provide therapeutic effects in a mouse model of the disease.

Methods : Four different truncated versions of the CEP290 coding sequence were packaged into AAV8 capsids. These vectors were injected subretinally into 14-day-old rd16/Nrl-KO double mutant mice modeling cone photoreceptor degeneration of CEP290-LCA. Electroretinography (ERG) and optokinetic tests were performed to monitor the retinal function and visual behavior of these mice. Immunoblot, immunofluorescence and electron microscopy (EM) were conducted to examine alterations of gene expression and retinal morphology following treatment. Protein-protein interaction was tested with co-immunoprecipitation. In-vivo gene knockdown was performed using AAV-delivered CRISPR -Cas9.

Results : A C-terminal CEP290 protein fragment expressed by the AAV vector was found to be effective in preserving the retinal function in rd16/Nrl-KO mice over an eight-month period. The mice exhibited a significantly improved optokinetic response in the vector-treated eyes than the control eyes. Following vector delivery, the C-terminal CEP290 protein fragment was localized to the connecting cilia of the photoreceptors and was able to maintain the normal expression and localization of a few CEP290-interacting cilia proteins. The treatment preserved a relatively normal structure of the connecting cilia and trafficking of the proteins involved in photo-transduction. In-vivo knockdown and co-immunoprecipitation studies revealed interdependency of the C-terminal CEP290 fragment and the mutant form of CEP290 in the mice for functional rescue.

Conclusions : Our study established that an LCA-causing CEP290 mutation can be complemented in-trans by a truncated version of the WT protein, which provides a new insight in developing therapies for diseases associated with CEP290 mutations.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.