Context Matters: The Importance of a Comprehensive Genomic Region When Assessing the Therapeutic Potential of Antisense Oligonucleotides in Splicing Assays.

Mini/midigene splicing assays are often used to evaluate splicing modulation therapy, for example, employing antisense oligonucleotides (AONs). Twenty-five AONs targeting the splicing defect caused by a recurrent variant in ABCA4 (c.768G>T) were tested using a midigene containing a part of intron 5, exon 6, and a part of intron 6 of the ABCA4 gene. Surprisingly, almost all AONs showed high efficacy, complicating candidate selection. We hypothesized that the lack of genomic context may lead to a very accessible transcript for AONs. Indeed, the use of an ABCA4 maxigene that contains a part of intron 5, exon 6, parts of intron 6, and the genomic region between exons 7 and 11 allowed a clear distinction between efficacious and less efficacious AONs, corroborating the results we recently observed in patient-derived retinal cells. These underscore the necessity of a proper genetic context included in constructs used in splicing assays to assess the potential of splicing modulation therapy.