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Yan Zhang, Lisa M. Baye, Qihong Zhang, John Beck, Xiaobai Patrinostro, Svetha Swaminathan, Diane C. Slusarski, Val Sheffield, Edwin M. Stone; A CEP290 N-terminal Protein Fragment Rescues A Zebrafish Model Of CEP290 Leber’s Congenital Amaurosis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1400.
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CEP290 is a large, multi-domain protein implicated in several cilia related syndromic disorders including Meckel-Gruber, Joubert, Senor-Loken and Bardet-Biedl Syndrome (BBS). Moreover, CEP290 is the most frequently mutated gene underlying the non-syndromic blinding disorder, Leber's congenital amaurosis (LCA). The purpose of the current study is to characterize the function of various CEP290 domains and to characterize a zebrafish model aimed at progressing towards future therapy for patients with CEP290 LCA.
We generated several truncated CEP290 protein fragments, which comprise several domains of the whole protein. We tagged these fragments with GFP and examined their localization in 293T and RPE cells. We also evaluated the physical interaction between these truncated proteins and other cilia proteins by co-immunoprecipitation. To determine the in vivo function of cep290 in the zebrafish, we examined the effects of cep290 knockdown using an antisense oligonucleotide (Morpholino, MO) designed to generate an altered cep290 splice product that models a common LCA mutation.
The N-terminal and C-terminal fragments of CEP290 both localize to the centrosome. The N-terminus interacts with NPHP2 and NPHP5, whereas the C-terminus interacts with MKS1. Histological analysis of the retina of cep290 knockdown zebrafish revealed no gross morphological defects; however, knockdown of cep290 results in a statistically significant reduction in visual function. We tested whether truncated CEP290 proteins could rescue the cep290 MO knockdown phenotype in zebrafish by co-injection of RNA with the cep290 MO. We demonstrate that vision impairment caused by disruption of cep290 can be rescued by expressing the N-terminus protein fragment.
These data reveal that a specific region of the CEP290 protein is necessary for vision and identify a potential gene therapy strategy for LCA patients with mutations in CEP290.
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