Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: gene transfer/gene therapy • gene/expression • photoreceptors