September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Patient-specific iPSCs to investigate pathophysiology and develop treatments for RPGR-associated XLRP
Author Affiliations & Notes
  • Joseph C Giacalone
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Erin R Burnight
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Tasneem P Sharma
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Luke A Wiley
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Dalyz Ochoa
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Malia Collins
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Robert F Mullins
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Budd A Tucker
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Edwin M Stone
    Department of Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Joseph Giacalone, None; Erin Burnight, None; Tasneem Sharma, None; Luke Wiley, None; Dalyz Ochoa, None; Malia Collins, None; Robert Mullins, None; Budd Tucker, None; Edwin Stone, None
  • Footnotes
    Support  Wynn Institute Endowment for Vision Research, Foundation Fighting Blindness (FFB)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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