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B. Chen, M. B. O’Donoghue, A. S. Lewin, M. S. Gorbatyuk; Testing RPGR Specific Ribozyme and siRNA in vitro: Tools for the Treatment of Dominant X-Linked Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5345. doi: https://doi.org/.
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X-linked retinitis pigmentosa (XLRP) caused by RPGR ORF15 mutations cause severe, early onset retinal disease, and these mutations may also affect female carriers leading to adult onset RP. Such a dominant effect might be reduced by suppressing the expression of RPGR-ORF15. A canine model (XLPRA2) exists for dominant XLRP caused by an ORF15 frameshift. Therefore, we constructed a new RPGR plasmid based on the dog RPGR sequence and tested the knockdown effect of a ribozyme and a siRNA in HEK293 cells.
We reconstructed the new RPGR plasmid pBL-sAP-RPGR from psiTEST vector (Invivogen) and the first 11 exons of the dog RPGR gene. We constructed plasmids containing a hammerhead ribozyme (Rz1273) expressed from the chicken β-actin promoter or a siRNA expressed as a small hairpin RNA from the H1 promoter. The ribozyme and the siRNA were designed to cleave at the same location in RPGR. These were co-transfected with pBL-sAP-RPGR in HEK293 cells and secreted alkaline phosphatase activity was measured in the culture supernatant in quadruplicate in order to estimate the knockdown effect.
The RPGR plasmid could effectively transfect HEK293 cells and lead to the secretion of alkaline phosphatase. HEK293 cells co-transfected with the sAP-RPGR plasmid and the ribozyme or siRNA at a ratio 1:5 showed a reduction of the RPGR expression by 89% and 57%, respectively.
The RPGR plasmid (pBL-sAP-RPGR) can provide a rapid, simple, and convenient method to screen for functional siRNA and ribozyme sequences in XLRP. Knockdown mutant RPGR gene transcripts may be a good gene therapy for dominant XLRP.
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