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Harry Otway Orlans, Michelle McClements, Maria In?s Patrício, Alun R Barnard, Robert E MacLaren; An optimized AAV vector for rod-specific gene expression. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4534.
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Many adeno-associated viruses (AAV) designed for the treatment of retinitis pigmentosa require a rod cell-specific promotor. Such promoters are often weaker than their ubiquitous counterparts and achieving adequate transgene expression given a limited packaging capacity remains a significant challenge. Here we describe a novel rod-specific human rhodopsin (RHOp)/chicken ß-actin/rabbit ß-globin (RhAG) hybrid promoter capable of achieving high levels of RHO expression that are equivalent to those attained using a self-complementary (sc) vector following subretinal delivery.
Four RHO-expressing AAV2/8 vectors were manufactured: single stranded (ss)RHOp.RHO with and without the woodchuck hepatitis post-transcriptional regulatory element (WPRE), ssRhAG.RHO.WPRE, and scRHOp.RHO. Their effect on RHO mRNA levels was compared in transduced dissociated retinal cells extracted from post-natal day (PND) 5 Rho-/- mice. To compare effects in vivo, 2x109 genome copies of each AAV were delivered subretinally in PND21 Nrl-GFP/Rho-/- mice. Four weeks later, photoreceptor layer (PRL) thickness was assessed using ocular coherence tomography (OCT). Rhodopsin protein levels were subsequently quantified by western blot densitometry and protein localization determined in tissue sections by immunohistochemistry (IHC).
No significant difference in RHO mRNA level was identified for dissociated retinal cells transduced with ssRHOp.RHO±WPRE and RhAG.RHO.WPRE. Transduction with scRHOp.RHO however resulted in a 4-fold increase compared with the other AAVs (p<0.01). Mean PRL thickness as measured by OCT was greater in scRHOp.RHO (39.8±1.6µm) and RhAG.RHO.WPRE (40.4±0.8µm) transduced eyes than those transduced with ssRHOp.RHO±WPRE (35.3±1.4µm & 36±1.5µm), p<0.05. Similar quantities of protein were obtained with scRHOp.RHO and RhAG.RHO.WPRE vectors and both achieved levels of rhodopsin 3-10x that of ssRHOp.RHO±WPRE (p=0.006 & 0.02 respectively). IHC revealed growth of outer segments packed with rhodopsin in transduced retinas but not in uninjected eyes. RHO was not detected in other retinal/RPE cells.
Inclusion of the exon/intron/exon sequence from the chicken ß-actin/rabbit ß-globin genes in the 5’-UTR of rhodopsin significantly increases AAV-mediated gene expression whilst maintaining cellular specificity. This construct achieved equivalent levels of protein to a self-complementary vector but with a much shorter AAV genome.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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