A new method to determine efficacies to treat RPGR mutations: Quantification of RPGR transport along the primary cilium in patient-derived cells

Abstract

Purpose: Retinal dystrophies constitute a group of clinically and genetically heterogeneous diseases. As treatments are not yet readily available for the majority of genetic eye diseases, read-out assays performed in patient-derived cells can aid in the development and comparative analysis of therapeutic approaches.

Methods: The studied patient carried a point mutation (RPGR: c.1245+3A>T), which led to skipping of RPGR exon 10. Patient-derived and control fibroblast cell lines were cultured. Formation of primary cilia was induced by serum starvation. Immunocytochemical detection of the RPGR protein provided the basis to localize and quantify RPGR along the primary cilium. A U7snRNA was modified and transduced into control cell lines to interfere with the normal transcript processing of RPGR. To silence the effect of the mutation, a mutation-adapted splice factor (U1 snRNA) was applied to patient-derived cells. Lentiviral shuttles were used to transduce primary cell lines.

Results: We describe the development of a new method with which the localization of the retinitis pigmentosa GTPase regulator (RPGR) protein along the cilium can be used as a measure for treatment efficacy. In a patient-derived fibroblast cell line, we found that the RPGR protein was retained in the transition zone of the cilium and lacked its localization along the axoneme. First, we aimed to confirm that the patient-specific exon skipping is causative for the aberrant ciliary localization and used an U7snRNA-based antisense approach to reproduce the patients' splice defect in control cells. This technology resulted in a 75% reduction in cilia with RPGR along the axoneme. Second, we aimed to treat the mutation-induced splice defect in the patient-derived fibroblasts using therapeutic U1snRNA. This resulted in a significant increase in the amount of the normal RPGR transcript and corrected the proteins’ localization at the cilium.

Conclusions: In this study, we show that detecting the RPGR protein along the cilium provides a reliable and quantifiable read-out assay to evaluate the efficacy of therapies intended to correct or silence RPGR gene mutations. This method opens the possibility to compare different therapeutic agents and thus, facilitates the identification of treatment options for the clinically and molecularly complex RPGR-associated diseases.