A single abbreviated ORF15 variant appears sufficient to reconstitute RPGR function in vivo

Abstract

Abstract: : Purpose: The retinitis pigmentosa GTPase regulator (RPGR) is required for maintaining photoreceptor viability. RPGR is expressed as constitutive and ORF15 variants due to complex alternative splicing. This study was designed to examine if the retina–specific ORF15 variant alone is sufficient for RPGR function. A further objective was to test if the highly repetitive "linker" region of ORF15 could be abbreviated without ablating the function, so that an RPGR replacement gene could be accommodated by AAV vectors. Methods: A cDNA representing RPGR–ORF15 but shortened by 534 bp in the linker region was placed under the control of a chicken beta–actin (CBA) hybrid promoter. The resulting construct was transfected into mouse embryonic stem (ES) cells. Clones expressing the transgene were selected and injected into mouse blastocysts. Transgenic chimeras were crossed with RPGR knockout mice. Offspring mice were studied from 1 to 14 months of age by histology, immunocytochemistry, electron microscopy and electroretinography (ERG). Results: One line of mice expressed RPGR uniformly in the connecting cilia of rod and cone photoreceptors, at approximately 30% of the WT level. Analyses of photoreceptor morphology, cone opsin mislocalization, upregulation of GFAP (a marker for retinal degeneration) and ERG were all consistent with the transgene exerting a substantial rescuing effect on the retinal degeneration phenotype caused by the loss of RPGR. Conclusions: RPGR–ORF15 is the functionally significant variant in photoreceptors. The length of its repetitive linker region can be reduced while still preserving function. Our findings should facilitate the design of replacement gene therapy for RPGR null mutations.

Keywords: retinal degenerations: hereditary • transgenics/knock–outs • gene transfer/gene therapy