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Tasneem P Sharma, Joseph C Giacalone, Erin R Burnight, Luke A Wiley, Jill S Wiley, Robert A Madumba, Robert F Mullins, Edwin M Stone, Budd A Tucker; HUMAN DISEASE MODELING OF TRNT1-ASSOCIATED RETINITIS PIGMENTOSA IN PATIENT-DERIVED CELLS. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6060.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is a heterogeneous group of monogenic disorders characterized by progressive death of light-sensing photoreceptor cells. We recently identified novel mutations in the tRNA Nucleotidyl Transferase, CCA-Adding 1 (TRNT1) gene that cause early-onset non-syndromic RP. The purpose of this study was to: 1) elucidate the pathophysiologic mechanism(s) of TRNT1 mutations in photoreceptor cell dysfunction and 2) rescue the functional deficit in TRNT1 by gene augmentation and CRISPR/Cas9 genome editing-based therapeutic approaches in patient-specific cells.
Dermal fibroblasts from patients with molecularly confirmed TRNT1-associated RP were expanded and targeted for induced pluripotent stem cell (iPSC) generation using OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed using standard rt-PCR, immunocytochemistry and TaqMan Scorecard Assay. Immunofluorescence and Western blot analyses were used to characterize expression of autophagy-specific proteins for disease modeling studies. Gene augmentation (AAV2/5-TRNT1) and CRISPR/Cas9-mediated genome editing studies using homology directed repair were performed to treat patient-specific hiPSCs-derived neural retinal cells and correct hiPSCs, respectively.
Mutations in TRNT1 caused reduced levels of full-length TRNT1 protein and expression of a truncated smaller protein in both patient-specific iPSCs and iPSC-derived neural retinal cells. Likewise, both patient-specific cell types exhibited a deficit in autophagic flux, as evidenced by aberrant accumulation of LC3-II. Gene augmentation (AAV2/5-TRNT1) restored a normal level of autophagic flux within iPSC-derived neural retinal cells. In addition, CRISPR/Cas9-mediated genome editing corrected the mutant phenotype and enabled generation of normal neural retinal cells.
Mutations in TRNT1 are capable of causing early onset, non-syndromic autosomal recessive RP. To model this disease in vitro, we have generated patient-specific iPSCs and iPSC-derived neural retinal cells. Furthermore, we have successfully investigated the pathophysiology of TRNT1-associated RP in this model and explored plausible therapeutic gene augmentation and genome editing-based approaches using patient-specific cells. Our autologous stem cell-based treatment strategy provides a foundation for future clinical trials in patients suffering from TRNT1-associated retinitis pigmentosa.
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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