Abstract
Purpose :
X-linked Retinitis Pigmentosa (XLRP) most often is caused by mutations in the exon ORF15 of the retinitis pigmentosa GTPase regulator (RPGR) gene. Since the majority of the mutations are point mutations, gene targeting using I-SceI endonuclease and classical gene therapy using silencing RNA (siRNA) may be used as treatment approaches. These strategies were tested in a cell culture model based on a XLRP mouse model (B6J.SV129-Rpgrtm1stie).
Methods :
The XLRP mouse model contains a pathologic mutation (del2793A), two silent mutations in the ORF15 exon and an I-SceI recognition site. A HEK293-ORF15mut cell line, containing the mutations identical to the B6J.SV129-Rpgrtm1stie mouse, was generated by spontaneous immortalization following pcDNA3.OFR15mut transfection. I-SceI cDNA sequence was cloned into a bicistronic expression cassette together with the GFP cDNA separated by a T2A linker sequence. The plasmid was transfected into the HEK293-ORF15mut cell line, GFP positive cells were sorted 48 h later by Fluorescent Activated Cell Sorting (FACS) and double strand break (DSB) repair events were analysed using the SURVEYOR assay. Different short hairpin RNAs (shRNAs) specific to the ORF15 sequence were created and the siRNA vector psiRNA4_7SKGFPzeo was used as backbone. The plasmids were transfected into the HEK293-ORF15mut cell line, GFP positive cells were sorted 24 h later by FACS. RNA was isolated and analysed by quantitative PCR (qPCR) for downregulation of RPGR-ORF15 expression.
Results :
Following transfection of the HEK293-ORF15mut cell line with the I-SceI-GFP plasmid, up to 55% of the FACS enriched GFP positive cells showed DSB repair activity compared to only 7% unenriched cells and no background in non-transfected cells. ShRNA transfection caused 57% downregulation of the RPGR-ORF15 transcript in HEK293-ORF15mut cells by the most efficient variant.
Conclusions :
Gene targeting and gene silencing are both promising strategies in vitro in stable cell lines. Both approaches will be analyzed in organotypic retinal cultures containing postmitotic cells for further optimization prior to enrolling the optimal approach in vivo in the mouse model of XLRP.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.