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Joseph C Giacalone, Erin R Burnight, Tasneem P Sharma, Luke A Wiley, Dalyz Ochoa, Malia Collins, Robert F Mullins, Budd A Tucker, Edwin M Stone; Patient-specific iPSCs to investigate pathophysiology and develop treatments for RPGR-associated XLRP. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis Pigmentosa (RP) is a heterogeneous disease that causes death of the light sensing photoreceptor cells of the outer neural retina and affects as many as 80,000 individuals in the US alone. X-linked RP (XLRP) is responsible for some of the most severe and earliest onset cases. The majority of XLRP cases are caused by mutations in the gene RPGR. The retinal-predominant isoform of RPGR, known as ORF15, contains a long repetitive sequence, which harbors the majority of the pathogenic mutations that cause RPGR-associated XLRP. The purpose of this study was to: 1) model RPGR-associated XLRP using patient-specific induced pluripotent stem cells (iPSCs) and 2) develop CRISPR/Cas9-based genome editing strategies for correction of disease-causing mutations.
Patient-specific iPSCs were generated from dermal fibroblasts of 7 patients with molecularly confirmed RPGR-associated XLRP. Pluripotency was confirmed using rt-PCR, immunocytochemistry and TaqMan Scorecard Assay. CRISPR/Cas9 constructs were generated to target patient-specific mutations. Gene targeting constructs and homology directed repair constructs were introduced to iPSCs via NEON transfection. Correction was confirmed via T7E1 assay and Sanger sequencing. Patient-specific iPSC-derived 3D retinal eyecups were generated from corrected and uncorrected cell lines and characterized via Western blot, immunocytochemistry and confocal microscopy.
Seven iPSC lines were generated with varying mutations, disease severity, and disease phenotypes. Genome editing of patient-specific iPSCs was achieved with transfection efficiencies of 30 percent, and the resulting modified iPSC clones were isolated and expanded via reporter selection. Patient-specific iPSC-derived retinal eyecups were generated and displayed the retinal progenitor and photoreceptor-specific markers PAX6, OTX2, RCVRN, CRX and NRL.
With the advent of iPSC technology, we are now able to generate retinal cells from male patients with mutations in RPGR ORF15. As a result, we have a unique opportunity to investigate patient-specific pathophysiologic mechanisms and therapeutic interventions in human cells. We have shown that genome editing via the CRISPR/Cas9 system can successfully correct patient-specific mutations in iPSCs. Moreover, corrected versus uncorrected lines will serve as a valuable tool for characterizing the observed disease phenotype.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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