Molecular mechanisms of disease in CEP290-related ciliopathies

Primary cilia act as cellular “antennae” that mediate diverse sensory roles. Primary ciliopathies are a group of inherited developmental disorders resulting from defects in the primary cilium. CEP290 is the largest individual protein in the primary cilium and is a key regulator of ciliary content and cilia formation. CEP290 mutations are the most frequent cause of autosomal recessive ciliopathies (incidence up to 1 in 15,000), that extend from severe syndromic neurodevelopmental disorders to congenital retinal dystrophy. The molecular basis for this phenotypic variability is unknown. A major research focus is therefore gaining mechanistic insight into the tissue-specific roles of CEP290 and variable pathogenicity of CEP290 mutations to enable development of therapeutics. This project used a functional genomics approach, using advanced 3D cell models of disease, to better understand the tissue-specificity of CEP290 function and CEP290 disease. Using RNAseq data, I described variable exon usage between tissues commonly associated with CEP290-related disease, indicating that alternative transcripts are important for tissue-specific roles of CEP290. 26/53 of the coding exons in CEP290 are in-phase “skiptic” (or skippable) exons. Variants in skiptic exons could be removed from CEP290 transcripts by skipping the exon, and nonsense-associated altered splicing (NAS) has been proposed as a mechanism that attenuates the pathogenicity of nonsense or frameshift CEP290 variants. I therefore used bioinformatic analysis of known pathogenic nonsense variants in CEP290 and identified a potential for splicing defects arising from exonic mutations, supporting the hypothesis that NAS accounts for pleiotropy in CEP290-related disease. Skiptic exon 36 was identified as a hotspot exon for isolated retinal dystrophies arising from nonsense variants in patients. I therefore modelled mutations in CEP290 exon 36 using CRISPR-Cas9 gene editing in induced pluripotent stem (iPS) cells and revealed a capacity for sequence-specific exon skipping. Finally, mutant iPS cells were differentiated to retina and kidney organoids. Analysis revealed, for the first time, tissue-specific exon skipping resulting from mutations in CEP290 exon 36. Importantly, exon 36 skipping had the capacity to ameliorate cellular disease phenotypes in these models. These findings are a strong indication that sequence- and tissue-specific altered splicing can explain the variable phenotypes of CEP290-related ciliopathies. This study provides a significant advancement in our understanding of CEP290-related disease pleiotropy. Use of advanced human 3D cell models has overcome limitations of previous studies by using physiologically relevant human tissue contexts. Future research should now be focussed on understanding the mechanisms of tissue-specific exon skipping and developing therapeutic splice-switching antisense oligonucleotides for pre-clinical studies.