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Alejandro Garanto, Frans Cremers, Rob Collin; Antisense oligonucleotide-based therapy for CEP290-associated LCA. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2737.
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© ARVO (1962-2015); The Authors (2016-present)
The most frequent genetic cause of Leber congenital amaurosis (LCA) is an intronic change in CEP290, that results in the insertion of a cryptic exon (exon X) into the CEP290 mRNA. Recently, we have shown promising results of antisense oligonucleotide (AON)-based therapy for this disorder, since transient transfection of AONs to cultured lymphoblastoïd cells of these patients almost completely rescued the aberrant splicing of CEP290 mRNA that is caused by the intronic mutation. The aim of this work is to further develop this therapeutic strategy, by employing other cell types, generate viral vectors that express AONs and by characterizing a CEP290 knock-in mouse model that carries part of the human CEP290 gene including the intronic mutation, specifically designed for this purpose.
Skin biopsies from LCA patients homozygously carrying the intronic CEP290 mutation allowed the generation of fibroblast cell lines. These fibroblasts were transfected with either naked AONs or AONs cloned into viral constructs. CEP290 transcripts were analyzed by RT-PCR, and CEP290 protein levels via Western blot analysis. In addition, cilium length was assessed by immunostaining. Two knock-in models have been generated, and initially characterized at transcriptional and morphological levels, by RT-PCR and immunohistochemistry. Animal work was performed according to the ARVO statement for the use of animals in ophthalmic and vision research.
Our data have proved the efficacy of the naked AONs in fibroblasts, restoring the correct splicing of the transcript. We are currently working on the validation of these results, by measuring CEP290 protein levels and cilium length in AON-treated fibroblasts. We are also testing viral constructs to further application in cell lines and animal models. The initial characterization of the humanized models at transcriptional level, has shown an unexpected splicing pattern in the humanized mice. This new splicing event results in the insertion of a second cryptic exon (Y) in part of the CEP290 transcripts. Ongoing work will determine the presence/absence of the human aberrant exon (X) and exon Y, as well as whether the splicing events are tissue or mouse-strain-specific.
Together, this work will be instrumental for the development of AON-based therapy for CEP290-associated LCA.
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