Antisense oligonucleotide-based restoration of ABCA4 splicing defects caused by deep-intronic mutations associated with Stargardt disease

Abstract

Purpose : Mutations in ABCA4 lead to Stargardt disease (STGD1), a maculopathy that affects approximately 1 in every 10,000 individuals worldwide. In the last years, we and others have found several deep-intronic mutations in ABCA4 that underlie STGD1. These variants lead to splice defects by introducing pseudoexons into the mature mRNA, disrupt the reading frame and are therefore predicted to result in a decrease of ABCA4 protein. Antisense oligonucleotides (AONs) are small RNA molecules that can bind complementary to the pre-mRNA and thereby redirect splicing. Here, we aim to employ AONs to correct splicing defects caused by deep-intronic mutations in ABCA4.

Methods : We have selected two previously reported deep-intronic variants with an unknown effect on splicing, c.4539+2001G>A and c.4539+2028C>T, and seven novel intronic variants identified after whole-gene sequencing of ABCA4. To assess the nature of the splice defects, fibroblast and iPSC-derived photoreceptor cells were employed. Alternatively, when patient-derived cells were not available, midigene constructs containing the region of interest were generated in order to elucidate the effect of the variant at the RNA level. Three AONs were designed for each target and their efficacy to redirect splicing was assessed by co-transfection with midigenes in HEK293T cells, or by direct delivery to patient-derived cells.

Results : The deep-intronic variants resulted in mRNA pseudoexon insertions. For each variant, several AONs were tested together with a sense oligonucleotide (SON) that served as a negative control. At least one AON for each target was able to redirect splicing and increased the levels of the wild-type ABCA4 transcript. In addition, we were able to correct the splicing defect caused by two neighboring ABCA4 mutations using the same AONs. We are currently testing whether the increase of wild-type ABCA4 mRNA after AON treatment also leads to elevated levels of ABCA4 protein.

Conclusions : Overall, our results showed the efficacy of AONs to rescue splice defects associated with several deep-intronic ABCA4 mutations. These data demonstrate the high therapeutic potential of RNA-based strategies to treat STGD1.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.