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Laura Moreno Leon, Linjin Li, Emma L West, Michelle O’Hara-Wright, Alexander J Smith, Robin R Ali, Samuel G Jacobson, Artur V Cideciyan, Hemant Khanna; Alterations in RPGR isoform levels play a critical role in X-Linked Retinitis Pigmentosa pathogenesis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1955.
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Mutations in RPGR are the most frequent cause of X-linked RP. Advances in retinal gene therapy have led to clinical trials of gene augmentation due to mutations in exon ORF15 of the RPGRORF15 isoform (terminating in intron 15). The RPGR gene is alternatively spliced and the role of the other major RPGR isoform, RPGRCONST (19 exons) in the maintenance of vision is still unclear. It is also not known whether mutations in exon ORF15 affect expression, stability or splicing of the RPGRCONST isoform. Our aim is to delineate the interplay between the RPGR isoform expression in regulating ciliary trafficking and its disruption due to mutations in RPGRORF15.
Fibroblasts were derived from skin biopsies of patients with distinct RPGRORF15 mutations or control individuals. Pluripotent stem cells were generated and differentiated to retinal organoids using standardized procedures. RPGR isoform expression was quantified by RT-qPCR and immunoblotting. Transcriptional regulation was determined by assessing transcript stability using Actinomycin-D treated fibroblasts and quantifying mRNAs levels by RT-qPCR in a time-dependent manner. Given that photoreceptor outer segments are modified sensory cilia and RPGR plays critical roles in ciliary trafficking and extension, immunofluorescence analyses were performed using marker antibodies to assess ciliary elongation and trafficking.
We found that some RPGRORF15 mutations lead to an increase in the RPGRCONST levels in patient fibroblasts and retinal organoids. Interestingly, the mutant RPGRORF15 transcripts are relatively unstable; however, increase in the levels of both isoforms was observed in the mutant fibroblasts. This increase is likely due to a positive feedback effect on overall RPGR gene transcription. The increase in RPGRCONST levels was correlated with ciliary elongation whereas RPGRORF15 overexpression reduced cilia length. We also found that the success of RPGRORF15 gene augmentation depended upon the RPGRCONST levels and ciliary elongation in the mutant fibroblasts.
Our study demonstrates that exon ORF15 mutations affect RPGRconst message levels and the ratio of RPGRORF15 to RPGRCONST affects cilia length. We suggest that therapeutic strategies of knocking down the RPGRconst isoform supplemented to RPGRORF15 gene augmentation should be explored for mutations resulting in perturbed RPGR isoform ratios.
This is a 2020 ARVO Annual Meeting abstract.
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