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Rob W. Collin, Jeannette Bennicelli, Mohammed Toure, Anneke I. den Hollander, Frans P. Cremers, Jean Bennett; Antisense Oligonucleotide (AON)-based Therapy For Cep290-associated LCA. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3324.
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© ARVO (1962-2015); The Authors (2016-present)
The most frequent mutation that causes Leber congenital amaurosis (LCA) (present in ~15% of LCA patients from Europe and the US) is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. We developed a novel gene therapy strategy for this mutation, by using an approach that is based on antisense oligonucleotides (AONs), small RNA sequences that are able to redirect normal splicing of aberrantly processed pre-mRNA molecules.
EBV-transformed lymphoblastoid cells of patients homozygously carrying the intronic CEP290 mutation, as well as stable HEK293-T cell lines carrying mutant CEP290 minigenes, were transfected with AONs directed against the aberrant exon that is incorporated in the CEP290 mRNA of LCA patients carrying the intronic CEP290 mutation. RT-PCR analysis was used to assess the amount of aberrantly spliced CEP290. Viral constructs with potential therapeutic AONs were generated for AAV-based delivery of transgenes to photoreceptor cells.
Naked AON molecules aiming to redirect normal CEP290 splicing were capable of significantly reducing the amount of aberrantly spliced CEP290, both in cultured patient lymphoblasts as well as in stable HEK293-T cells carrying CEP290 minigenes. Potential therapeutic AON sequences were cloned into viral vectors carrying inverted terminal repeats for AAV synthesis, and a modified U7 snRNA that drives AON expression to the spliceosomal compartment in the nucleus. These constructs were also capable of redirecting normal CEP290 splicing in cultured cells.
AON-based therapy is a promising therapeutic approach for CEP290-associated LCA, as was shown in cultured cells. To continue this project, we have generated a transgenic knock-in mouse that carries part of the human CEP290 gene, including the complete intron harboring the frequent mutation, and are starting to characterize these animals to determine the therapeutic potential of AON-based therapy for CEP290-associated LCA in vivo.
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