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Rob W J Collin, Mubeen Khan, Riccardo Sangermano, Sarah Naessens, Miriam Bauwens, Carel C B Hoyng, Frauke Coppieters, Silvia Albert, Michael E Cheetham, Elfride De Baere, Frans P Cremers, Alex Garanto; Splice modulation therapy for a variety of ABCA4 mutations underlying Stargardt disease. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3401. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Antisense oligonucleotides (AONs) are small DNA/RNA molecules able to modulate pre-mRNA splicing, including the correction of aberrant splicing processes due to genetic mutations. Our proof-of-concept work on the correction of a recurrent splicing defect underlying CEP290-associated Leber congenital amaurosis (LCA) has led to the initiation of a clinical trial, with recently announced promising results. Here, we expand the use of AONs for the treatment of Stargardt disease, a retinal disorder caused by ABCA4 mutations.
We established a panel of ABCA4 midigenes together encompassing the complete ABCA4 gene, with which the effect of any ABCA4 variant on pre-mRNA splicing can be assessed in vitro. Following site-directed mutagenesis, wild-type or mutant midigenes were transfected into HEK293T cells to assess variants affecting ABCA4 pre-mRNA splicing. For variants resulting in aberrant ABCA4 splicing, AONs were designed, and co-transfected with the midigenes to assess their efficacy to correct splicing defects. For several variants, patient-derived fibroblasts and/or induced pluripotent stem cell (iPSC)-derived photoreceptor progenitor cells (PPCs) were treated with the AONs, and rescue efficacy was determined by RT-PCR analysis and Western blot analysis.
We identified more than a dozen different ABCA4 variants that affect pre-mRNA splicing. The majority of variants resides in introns, and leads to the insertion of pseudoexons (PEs) that are predicted to result in loss of ABCA4 protein function. Another, recurrent, ABCA4 variant (c.768G>T) led to an exon extension of 35 nucleotides (nt), also disrupting the reading-frame. For all variants that resulted in PE insertion, this process could be corrected by one or more AONs specifically targeting the corresponding PE. Intriguingly, also the 35-nt extension caused by the common c.768G>T variant could be rescued completely by blocking the alternative splice donor site in intron 6, redirecting the spliceosome to use the original splice donor site of exon 6.
AONs appear to be an effective and versatile tool to correct different types of splice defects caused by ABCA4 mutations. Given the promising data obtained so far in a clinical trial using AONs for the treatment of CEP290-associated LCA, AONs may serve as a more broadly applicable therapeutic strategy, not only for Stargardt disease but potentially also for other retinal diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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