AAV transfer of the human visual pigment core LCR region impacts the efficiency and specificity of in vivo photoreceptor expression.

Abstract

Abstract: : Purpose: The locus control region (LCR) adjacent to the red and green visual pigment gene array in human is essential for correct expression. We evaluated whether the conserved 37 bp core element of this LCR (cLCR) is a general enhancer element and defines expression specificity. Methods: Tandem cLCR elements, 2, 3 or 5 copies, were engineered in opposite orientations upstream of: 1) the –490/+6 fragment of human red promoter (Chop500), or 2) –585/+38 fragment of the RPE–specific VMD2 promoter (VMD2pro). The constructs, which included a GFP–reporter gene downstream the promoter region, were packaged into serotype 1 or 2 AAV capsids. Two µl of vector (2exp13 particles per ml) were injected subretinally into male Sprague–Dawley rats at P40–48. At PN 100–130 immunocytochemistry was employed to analyze patterns and efficiency of GFP reporter expression. Some retinas were collected and processed for real–time PCR to quantify the effect of multiple cLCR copies on transcriptional efficiency. Results: Without a LCR element AAV.Chop500.GFP expresses specifically but weekly in cone photoreceptors. In contrast, AAV.cLCR.Chop500.GFP (2, 3 or 5 cLCR copies) resulted in strong, photoreceptor–exclusive GFP expression. Both cones and rods were targeted. The transduction efficiency was greatly dependent on cLCR copy number with the 3– and 5–copy vectors being approximately10 times stronger than the 2–copy vector. Transduction did not depend on cLCR orientation in vector. Real– time PCR data confirmed the increased transcriptional efficiency as a function of cLCR copy number. Without a cLCR element AAV.VMD2pro.GFP expresses strongly and specifically in RPE cells. Placing multiple cLCR copies in front of the VMD2 promoter generated non–RPE specific expression, with primarily photoreceptors being targeted in addition to RPE cells when the vector is delivered to the subretinal space. Conclusions: Tandemly arrayed human opsin cLCR elements in an AAV vector greatly impact the cell–type expression profile of the vector. The cLCR appears to act as a potent orientation independent general enhancer with an additional ability to allow non–photoreceptor–specific promoters (e.g. the VMD2 promoter) to express in photoreceptors.

Keywords: gene/expression • photoreceptors • gene transfer/gene therapy