The importance of having a relevant model system to investigate splicing modulation in the retina: The unforeseen cases of the intronic variants in RPE65 and IMPG2 genes

Abstract

Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous disorders that can be caused by pathogenic variants in one of the more than 270 genes associated with IRDs. Among them, mutations in RPE65 can lead to Leber congenital amaurosis (LCA) whereas mutations in IMPG2 to vitelliform macular dystrophy (VMD).During my talk, I will provide evidence of the importance of employing the right cellular models to identify disease mechanism(s) and eventually evaluate suitable treatments. For this, I will highlight two intriguing examples of potential splicing defects studied by our group. Such defects are attractive for splicing modulation therapies using antisense oligonucleotides (AONs), a group of versatile molecules that can alter splicing by interacting with the target sequence(s) at pre‐mRNA level. Thus, characterizing the effect of a variant at the RNA level is instrumental to assess the applicability of AON‐based therapeutic intervention.We explored the effect of the homozygous variant c.11 + 5G>A in RPE65 and a heterozygous complex allele consisting of c.3023‐15T>A and c.3023G>A (p.(Gly1008Asp)) in IMPG2. In silico predictions suggested that these variants lead to a splicing defect. To assess the pathogenic effect of these variants, splice reporter assays using midigenes were conducted in HEK293T cells. Initial results pointed towards a 124‐nt exon elongation for the RPE65 variant and an in‐frame deletion of 48‐nt at the beginning of exon 15 for the complex allele in IMPG2.To validate these results, blood cells from the patients harbouring these defects were reprogrammed to induced pluripotent stem cells (iPSCs). Subsequently, iPSCs were differentiated to the retinal cells in which these genes are endogenously expressed. For RPE65, iPSCs were differentiated into retinal pigment epithelium (RPE) cells (>90 days differentiation) or, for IMPG2, into photoreceptor precursor cells (PPCs, 30 days differentiation). For the RPE65 variant, a cell specific effect was observed: while a clear exon elongation was observed in the midigene system, in RPE, the expression of the gene was highly reduced. In addition, no protein was detected and the patient‐derived cells showed differences compared to the control line. For IMPG2, the 48‐nt deletion was detected in a lower percentage in patient‐derived PPCs compared to the levels observed in the midigene system. However, aberrantly spliced IMPG2 transcripts were highly elevated in patient‐derived PPCs in comparison to control PPCs.Overall, our work shows the importance of having the right cellular context to determine the effect of genetic variants on splicing in the retina.