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Joseph Giacalone, Luke A Wiley, Luan A. Streb, Heather T. Daggett, Erin R Burnight, Robert F Mullins, Budd Tucker, Edwin M Stone; RPGR ORF15: From Clinical Sequencing to Treatment. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3597. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis Pigmentosa is a genetically heterogeneous disorder characterized by progressive photoreceptor degeneration. In X-linked Retinitis Pigmentosa (XLRP), a large number of disease causing mutations reside within the gene, RPGR. Interestingly, RPGR undergoes alternative splicing, and one variant, containing ORF15, has a significant role in XLRP. Specifically, the 3 prime end of the ORF15 transcript is a highly repetitive, purine rich region that is susceptible to mutations. To date, we have screened 152 families using a cloning-based sequencing technique, which resulted in 27 positive families with 45 affected individuals, 13 of whom are female. In a subset of 15 patients with extensive clinical information, the age at diagnosis ranged from 4 to 48 years. Given the clinical impact of this mutational hotspot, treatment options are urgently needed.
Skin biopsies were obtained from molecularly confirmed XLRP patients with terminating variants residing within the ORF15 region of RPGR. Patient-specific iPSCs were generated from dermal fibroblasts. Adeno-associated viral (AAV) constructs carrying the ubiquitous form of RPGR and a non-ORF15 retinal-specific isoform under the control of the cytomegalovirus promoter were cloned, packaged and transduced into iPSC-derived retinal cells. CRISPR/Cas9 constructs targeting the ORF15 region and wild-type donor homology directed repair constructs were cloned.
The ubiquitous form of RPGR and a retinal specific isoform transduce iPSC-derived retinal neurons and drive full-length and retinal specific RPGR expression as shown by rt-PCR, immunoblotting and immunohistochemistry. Specific and efficient small guide RNA targeting of ORF15 modified specific RPGR locus was achieved in HEK293t cells. A combined CRISPR/CAS and homology directed repair approach in patient-specific iPSCs and iPSC-derived retinal precursor cells was performed.
Terminating variants within the ORF15 have the potential to produce severe clinical phenotypes. The recent advances in the fields of gene therapy and genome editing present potential avenues for treating the diversity of cases observed. We have shown that genome editing via the CRISPR/Cas9 system can successfully target ORF15. Additionally, gene therapy may present another means of treatment.
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