Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease

Abstract

Stargardt disease is a progressive retinal disorder caused by biallelic mutations in the ABCA4 gene that encodes the ATPbinding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

The role of Musashi-1 in CEP290 c.2991+1655A>G cryptic exon splicing in Leber Congenital Amaurosis

“…In particular, the brain shows the most abundant degree of AS and has been under the spotlight since the pioneering studies on microexons and their implication in autism (Irimia et al, 2014). The retina, as part of the CNS, has recently come into focus and an increasing body of evidence points to genetic variants affecting AS associated with inherited blinding conditions; namely, ABCA4 , CEP290 , DYNC2H1 (Khan et al, 2020; Parfitt et al, 2016; Vig et al, 2020).…”

“…However, this might be because many AS events we identified share a broader neuronal expression, rather than an exclusively retinal one, such that the link with CEP90-LCA is still missing. Nonetheless, iPSC-derived ROs are a valuable model to faithfully recapitulate conditions affecting AS in human retina, with special relevance to intronic variants and for developing potential therapies (Cideciyan et al, 2019, 2021; Khan et al, 2020; Parfitt et al, 2016).…”

“…Overall, these studies support the view that human photoreceptors maximise alternative exon splicing, leading to a highly specialised transcriptome during development and differentiation. This raises the possibility, however, that the retina could be particularly vulnerable to intronic mutations, compared to other tissues, as observed for the CEP290-LCA variant (Parfitt et al, 2016) and for variants within the introns of ABCA4 that lead to Stargardt Disease (Khan et al, 2020). The mRNA processing mechanisms causing this retinal sensitivity to deep intronic variants resulting in aberrant splicing are still unknown.…”

The Rise of Retinal Organoids for Vision Research

“…To replicate the RPE65 -associated LCA and AlPL1 -LCA phenotype in a 3D in vitro system, patient-specific iPSCs were generated that were later used to generate organoids [ 62 , 63 ]. A similar study explored pathogenic splicing variants of the ABCA4 gene, a transporter protein responsible for Stargardt’s disease [ 64 ]. These studies have emphasized the importance of patient-specific, disease-targeted 3D model systems.…”

Inducible Pluripotent Stem Cells to Model and Treat Inherited Degenerative Diseases of the Outer Retina: 3D-Organoids Limitations and Bioengineering Solutions

“…Furthermore, phenotypes of mutations in the CRB1 gene, leading to disruption of the OLM [ 52 ] (see Section 3.1 ) and in the RS1 gene, causing X-linked juvenile retinoschisis [ 53 ], have also been recapitulated in organoids derived from specific patients. Additional models include adRP [ 54 ], non-syndromic RP [ 44 , 49 , 55 , 56 , 57 ], and LCA [ 51 ].…”