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Jean-Michel Rozet, Xavier Gerard, Isabelle Perrault, Eduardo Silva, Karine Bigot, Sabine Defoort-Delhemmes, Arnold Munnich, Daniel Scherman, Antoine Kichler, Josseline Kaplan; Antisense Oligonucleotide-mediated Exon Skipping Improves Primary Cilia Assembly In Fibroblasts Harbouring The Common Lca Cep290 C.2991+1655g>A Mutation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1920.
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Leber congenital amaurosis (LCA) is a severe hereditary retinal dystrophy responsible for congenital or early-onset blindness. The most common disease-causing mutation (>10%) is located deep in intron 26 of the CEP290 gene (c.2991+1655 A>G) where it creates a strong splice donor site that leads to the insertion of a cryptic exon encoding a premature stop codon. The aim of this study was to assess the feasibility of an antisense oligonucleotide (AON)-mediated exon skipping strategy to correct this aberrant splicing.
Fibroblast cell lines of patients harbouring the c.2991+1655 A>G mutation (n=4, 3/4 homozygous) and controls (n=3) were transfected using antisense and sense 2’O-methyl phosphorothioate-modified oligonucleotides designed to target exon splicing enhancer (ESE) around the mutation. The skipping was optimized for oligonucleotide sequences and concentrations, transfection conditions and treatment time. The efficiency of skipping was assessed using qRT-PCR, Western blot analysis using a polyclonal antibody recognizing the C-terminus of the CEP290 protein and primary cilia counting.
The level of expression of CEP290 messengers was unchanged when control cell lines were transfected using the sense ONs or AONs (p>0.05). Likewise, no change in expression was noted when patient’s cells were treated with the sense ONs (p>0.05). Conversely, a highly significant increase in expression of the wildtype CEP290 allele was obtained when cells were treated with AONs (0.029<p<0.002) with expression levels reaching that of controls. Western blot analysis evidenced increased levels of CEP290 in patients’ cell lines treated with the AONs but not the sense ONs. Finally, following serum-starvation, primary cilia expression was significantly reduced in patient’s fibroblasts compared to controls lines (48.6%± 6.5% vs 83.6%±3.2 %; p=0.0097). Upon transfection with the antisense ONs but not the sense versions, the proportion of ciliated cells increased significantly in patients, reaching levels similar to controls (75.3%±3.5% vs 78.3%±3.4%; p=0.624).
Our results suggest that antisense ON-mediated exon skipping resulted in a significant improvement of cilia assembly and/or maintenance.CEP290 mutations are the most common cause of LCA. The results of this study show therapeutic potential of exon skipping for the treatment of the mutation c.2291+1655A>G which alone accounts for 10% of LCA cases.
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