Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells.

Alexander G Bassuk,Yao Li,Vinit B Mahajan,Andrew Zheng,Stephen H Tsang

doi:10.1038/srep19969

Abstract

Induced pluripotent stem cells (iPSCs) generated from patient fibroblasts could potentially be used as a source of autologous cells for transplantation in retinal disease. Patient-derived iPSCs, however, would still harbor disease-causing mutations. To generate healthy patient-derived cells, mutations might be repaired with new gene-editing technology based on the bacterial system of clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9, thereby yielding grafts that require no patient immunosuppression. We tested whether CRISPR/Cas9 could be used in patient-specific iPSCs to precisely repair an RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce iPSCs carrying the patient’s c.3070G > T mutation. The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene’s repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal disease.

Highlights

Stem cell-derived cell transplantation in the eye is one therapy being explored for inherited retinal degenerations such as retinitis pigmentosa (RP)
We reported previously on the diagnosis of retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP) in two brothers with significant vision loss[6]
Whole exome sequencing with Sanger sequencing confirmation uncovered a novel RPGR mutation (c.3070G > T, pGlu1024X) within the open reading frame-15 (ORF15) exon[6], a lengthy and repetitive, purine-rich region of the gene that accounts for > 60% of all XLRP mutations[7]

Summary

Introduction

Stem cell-derived cell transplantation in the eye is one therapy being explored for inherited retinal degenerations such as retinitis pigmentosa (RP). Recent clinical trials evaluating allogeneic retinal grafts derived from human embryonic stem cells (hESCs) show the procedure to be safe and potentially effective[1]. Using a patient’s own cells for transplantation would avoid these pitfalls and is possible with induced pluripotent stem cells (iPSCs). Through established protocols[2], fibroblasts from a skin biopsy can be returned to a pluripotent state and serve as a renewable, autologous source of replacement cells that avoids the ethical complications of hESCs. the original disease-causing mutation would still be present in patient iPSCs, precise mutation correction is possible through gene editing techniques adapted from the bacterial clustered regularly interspersed short palindromic repeats (CRISPR)/Cas[9] system[3]. We tested whether CRISPR could precisely edit the pathogenic mutation and produce gene-corrected iPSCs for eventual use in autologous transplantation

Methods

Results

Conclusion