Fig. 3: Codon modification of human rhodopsin.

a Dual Glo^® assays demonstrate codon-modification of target regions confers resistance to suppression by the corresponding mirtron(s). Mean ± SEM plotted, n = 6. ****p < 0.0001, two-sided Dunnett’s multiple comparisons test. RHO^M3R: M3-resistant RHO; RHO^M5R: M5-resistant RHO; RHO^M3/5R: M3- and M5-resistant RHO. b Rhodopsin immunostaining of wild type RHO- and codon modified RHO^M3/5R-transfected HEK293 cells (n = 3). Scale bar = 250 µm. c High magnification view confirming RHO and RHO^M3/5R traffic in a similar fashion when expressed in HEK293 cells. d ERG confirms functionality of human transgenic rhodopsin protein expressed from the codon modified RHO^M3/5R transcript following subretinal delivery of AAV-M3.M5^H.RHO^M3/5R in rhodopsin-null mice. Dark-adapted a- and b-wave responses of paired uninjected (left) and injected (right) eyes (n = 10). A-wave flash intensity: 10 cd.s/m² representing a mixed rod-cone response. b-wave responses followed dim flashes of 0.0001 cd.s/m² and were thus predominantly rod-derived. ***p = 0.0003, ****p < 0.0001, two-sided Šidák’s multiple comparisons test. e Transgenic human rhodopsin is expressed in rods of Nrl.GFP/+, Rho^−/− mice following subretinal injection of AAV-M3.M5^H.RHO^M3/5R. Retinal cryosections from injected and uninjected eyes of an individual mouse stained for rhodopsin. n = 6. Scale bar = 200 µm. IPL: inner plexiform later, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segments, OS: outer segments. f High magnification section of an injected retina double stained for rhodopsin and cone arrestin. No overlap of staining was detected confirming restriction of transgenic protein expression to rods. The IS/OS of a single cone stained for cone arrestin (solid arrows) located between two rod OS expressing transgenic human rhodopsin (open arrows) is shown, suggesting rod-specific expression. Scale bar = 20 µm.