Expression of GFP and Edn2 from subretinally injected scAAV5.
(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-Gfp at PN8 and evaluated at PN10, PN12, and PN14 by GFP immunofluorescence (A, Panels 1–4). Significant GFP staining was not observed until PN12, with stronger staining at PN14, especially in the vicinity of the subretinal injection site (arrow). The spatial expression of GFP in WT retinas injected with scAAV5-smCBA-Gfp was also evaluated in paraffin sections at PN12 (A, Panel 5). GFP expression was observed predominantly in the ONL and RPE; sporadic expression of GFP in Müller cells was also observed in paraffin sections. (Bar = 25 µm.) (B) Schematic of the EDN2 cleavage events required to produce the mature EDN2 peptide. EDN2 is first produced as prepro EDN2 (175 aa) which is rapidly processed by furin-like endopeptidases to yield big EDN2 (38 aa). Big EDN2 must then be cleaved by an endothelin-specific converting enzyme (ECE) to produce the 21 a.a. mature EDN2 peptide that can bind to endothelin receptors (figure adapted from ). The regions of the Edn2 mRNA corresponding to the cDNAs cloned into the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 vectors are shown. (C) Expression of scAAV5-derived Edn2 mRNA in Pde6brd1/rd1 retinas at PN12 after injection of the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 constructs at PN8. scAAV5-derived Edn2 mRNA expression values are shown relative to the levels of endogenous Edn2 mRNA (from the same retina) and all values were normalized to Gapdh. scAAV5-preproEdn2 transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous Edn2 mRNA, while scAAV5-matEdn2 transcripts increased between 2.5 to 11.3-fold over the endogenous Edn2 mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.