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Alejandro Garanto, Lonneke Duijkers, Ru Xiao, Luk H Vandenberghe, Rob W J Collin; AAV-mediated antisense oligonucleotide-based therapy for CEP290-associated LCA. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3312.
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Leber congenital amaurosis (LCA) is a severe form of retinal degeneration that can be caused by mutations in more than 20 different genes. The most recurrent causative mutation is an intronic change in CEP290 (c.2991+1655A>G), that results in the insertion of a cryptic exon into CEP290 mRNA. Antisense oligonucleotides (AONs) are small RNA molecules able to redirect pre-mRNA splicing of specific targets. Recently, we have shown promising results employing AON-based therapy in cultured lymphoblastoid and fibroblast cells of LCA patients homozygously carrying the intronic CEP290 mutation. Correct splicing, ciliation and cilium length were almost completely rescued, and CEP290 protein levels significantly increased after AON treatment. The aim of this work is to further develop this therapeutic strategy, by employing viral vectors that express these AONs.
Different AONs were cloned into AAV vectors and their efficacy was assessed by co-transfecting the different AON together with a mini-gene containing the intronic mutation.
RT-PCR analysis revealed two highly effective AON-containing AAV-vectors that were able to redirect normal CEP290 pre-mRNA splicing. Currently, we are analyzing the tropism of several AAV serotypes to determine the most potent one for transducing fibroblast cell lines. Subsequently, we will generate AON-containing AAVs to assess their therapeutic efficiency by RT-PCR, immunodetection of CEP290 protein levels, ciliation and cilium length in patient’s fibroblast cells.
Herewith, we demonstrate the therapeutic potential of AAV-based delivery of AONs to treat CEP290-associated LCA.
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