A human-specific RPGR isoform and a clinically approved Rho/ROCK inhibitor ameliorate defects associated with RPGR dysfunction

Pathogenic variants in the <i>RPGR</i> gene are the primary cause of photoreceptor degeneration in X-linked retinitis pigmentosa (RP). Previous studies have linked <i>RPGR</i> dysfunction to defects in ciliary structure and actin turnover. <i>RPGR</i> encodes three major isoforms-<i>RPGR</i> ^<i>1-19</i> , <i>RPGR</i> ^<i>ORF15</i> , and the human-specific <i>RPGR</i> ^{<i>s14/15</i>} -yet the function of <i>RPGR</i> ^{<i>s14/15</i>} remains poorly understood. There is an urgent unmet need for effective treatments targeting <i>RPGR</i>-associated RP. We generated <i>RPGR</i> mutant hTERT-RPE1 cell lines and found that the loss of all <i>RPGR</i> isoforms resulted in pronounced ciliary defects, including aberrant cilia length and segmentation, along with disrupted actin turnover. Strikingly, cells expressing only the human-specific isoform <i>RPGR</i> ^{<i>s14/15</i>} closely resembled wild-type controls and were largely protected from these defects, underscoring a critical role for <i>RPGR</i> ^{<i>s14/15</i>} in maintaining ciliary integrity and actin dynamics. Furthermore, we demonstrated that pharmacological disruption of actin polymerization with cytochalasin D (CytoD) in control cells mimicked the ciliary abnormalities seen in <i>RPGR</i>_KO cells. Conversely, treatment with ripasudil-a clinically approved Rho/ROCK inhibitor-rescued both ciliary and actin-related defects in <i>RPGR</i>_KO lines without observable cellular side effects. In summary, our findings highlight the therapeutic relevance of the human-specific isoform <i>RPGR</i> ^{<i>s14/15</i>} and identify ripasudil as a promising candidate for treating <i>RPGR</i>-associated retinal degeneration.