GFP expression in the retina after the intravenous delivery of scAAV9-GFP in adult mice.

<p>Retinal cross sections were treated for GFP immunofluorescence (green) and counterstained with DAPI (blue), four weeks after the injection of 2×10¹² vg of scAAV9-GFP into the tail veins of eight-week-old mice. GFP was detected in all retina layers (A–D) and in the ciliary bodies (CB in A). Transduction efficiency was particularly high in the RGC layer (B–C) but GFP was also expressed in the various cell types of the inner nuclear layer (INL), including cells with the morphology of bipolar cells (arrowheads in C) and of Müller cells (arrows in D). Rare GFP-positive photoreceptors (asterisks in B and D) and RPE cells (arrowheads in D and F) were also detected. (E–G) High magnification of GFP-positive (E) Müller cells, (F) RPE cells and (G) photoreceptors. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion cell layer. Scale bar: 200 µm in A and B; 70 µm in C; 50 µm in D; 20 µm in E–G. A, B and E–G are epifluorescence images; C and D are confocal images.</p

GFP expression in RGC and bipolar cells.

<p>Representative retina sections from adult mice treated for double-immunofluorescence analysis four weeks after the injection of 2×10¹² vg of scAAV9-GFP vectors into the tail vein. (A–C) Double-labeling of transduced RGCs for GFP (green) and Brn-3a (red) (arrowheads indicate double-labeled RGCs and arrows indicate cells expressing GFP only). A transduced photoreceptor is highlighted with an asterisk. (D–F) Double-labeling of bipolar cells for GFP (green) and Chx10 (red). The arrow indicates a Chx10-positive bipolar cell with high levels of GFP. The retinal nerve fiber layer (RNFL), labeled for GFP due to the transduction of upstream RGC, is indicated by an asterisk. No RGC can be seen on this panel which illustrates the central part of the retina (as demonstrated by the thickness of the RNFL). ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion cell layer. Scale bar: 50 µm.</p

GFP expression in the optic nerve and the ciliary body of intravenous scAAV9-GFP injected adult mice.

<p>Representative cross sections of the optic nerve (A) and the ciliary body (B) treated for GFP immunofluorescence (green) and stained with DAPI (blue), four weeks after the injection of 2×10¹² vg of scAAV9-GFP into the tail veins of eight-week-old mice. In (A), the boundaries of the retinal nerve fiber layer (originating from the RGC) are clearly demarcated by their pattern of GFP expression (arrowheads) (arrows: GFP-positive axons in the optic nerve). (B) High magnification of the ciliary body, showing strong GFP expression in the epithelial cells. ON: optic nerve; RET: retina; TM: trabecular meshwork. Scale bar: 100 µm.</p

Systemic injection of AAV serotype 2 does not lead to transduction of the neural retina.

<p>GFP expression in representative cross-sections of the retina of adult mice one month after systemic administration of 2.10¹² vg scAAV-GFP of serotype 9 (A–F) or serotype 2 (G–L) in adult mice (n = 3 per condition). GFP expression was detected in the neural retina in all mice from the serotype 9 treated-group (panel A to F are from three different animals). As expected, the highest transduction efficiency was observed at the level of the RGC layer. In contrast, no GFP expression was detected in the retina after AAV serotype 2 injection (panels G to L are from three different animals). Left panels: GFP immunofluorescence; right panels: merged view of GFP (green) and dapi (blue). ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion cell layer. Scale bar: 100 µm.</p

The LV-RPE65 Vector Restores the ERG Response of the <i>Rpe65</i>^−/− Mice

<div><p><i>Rpe65</i>^−/− mice were treated P5 by an intravitreal injection of 20 ng of lentiviral vector.</p> <p>(A) ERG response to single flashes was recorded in scotopic condition at 2 mo of age. After LV-GFP treatment, the mice showed a response typical for <i>Rpe65</i>^−/− mice, with the a-wave absent and the b-wave elicited only for the highest stimuli (i.e., 10 and 25 cd s/m²; left tracings). After LV-RPE65 treatment, a stimulus of 10⁻² cd s/m² was sufficient to trigger the b-wave, and the a-wave appeared for a stimulus of 0.3 cd s/m² (right tracings). Vertical bars indicate occurrence of the stimuli; x-axis: 40 ms/div; y-axis: 100 μV/div.</p> <p>(B) Summary of the ERG recordings performed 2 mo after vector delivery at P5 in <i>Rpe65</i>^−/− mice. Amplitude of b-wave (expressed in μV) is represented for each stimulus intensity. In the LV-RPE65-treated group (squares), the b-wave rose with a stimulus as low as 10⁻² cd s/m², whereas in the LV-GFP-treated group (triangles) 3 cd s/m² were necessary to elicit the b-wave. ANOVA for repeated measures showed significant stimulus intensity effect (<i>p</i> < 0.0001), group effect (<i>p</i> = 0.023), and group versus stimulus intensity interaction (<i>p</i> = 0.002). Data are presented as mean ± standard error of the mean (SEM).</p> <p>(C) Improvement of the ERG response after LV-RPE65 treatment is correlated with the level of RPE65 expression. The area of the retina expressing GFP (triangles) or RPE65 (squares) was quantified for each eye at age 4 mo after gene transfer at P5, and plotted against the b-wave threshold (defined as the intensity of the lowest stimulus capable of eliciting the b-wave, expressed in log mcd s/m²).</p></div

S- and M/L-Cone Opsin Labelling Increased after LV-RPE65 Treatment of <i>Rpe65</i>^−/− Mice at P5

<div><p>(A) Double immunostaining of wild-type retina with antibodies directed against S-opsin (red) and M/L-opsin (green) localized in the outer segment. Note that some cones express both opsins while others are mainly labelled for only one of these opsins.</p> <p>(B–D) LV-RPE65 treatment allows similar expression of both S- and M/L-opsin to the wild-type retina in the region expressing the <i>Rpe65</i> transgene. Arrows indicate double labelling of cone opsins; arrowheads indicate cone stained only for S-opsin.</p> <p>(E and F) LV-GFP treatment does not rescue S- or M/L-opsin expression and shows a pattern of staining similar to untreated <i>Rpe65</i>^−/− (unpublished data). *Mislocalized and reduced S- and M/L-opsin expression.</p> <p>(G) Quantification of the three eyes with the highest transgene expression for LV-RPE65 and LV-GFP groups shows a significant increase in both S- and M/L-opsin expression after LV-RPE65 treatment compared to the LV-GFP (<i>p</i> = 0.005 for M/L-opsin and <i>p</i> < 0.0001 for S-opsin) or untreated (<i>n</i> = 4, <i>p</i> = 0.024 for M/L-opsin and <i>p</i> < 0.0001 for S-opsin) groups (*<i>p</i> < 0.05). No statistical difference was noted between LV-GFP and untreated knockout animals.</p> <p>Data are presented as mean ± SEM. Scale bar in (F): 500 μm in A; 50 μm in B–F. KO, <i>Rpe65</i>^−/− mice; LV-GFP, LV-GFP-treated mice; LV-RPE65, LV-RPE65-treated mice; WT, <i>Rpe65</i>^+/+ mice.</p></div

Retinal layer thickness measures in C57BL/6JRj wild-type mice by SD-OCT and histology.

<p>Retinal thickness in nasal and temporal sides in SD-OCT image (A) and in corresponding histological section (B). (C) Measures of retinal layers thickness by SD-OCT and histology in C57BL/6JRj mice, n = 11, Mann Whitney test. (D) Retinal thickness evaluated by SD-OCT and histology in C57BL/6JRj mice. Each pair of point represents the whole retina thickness of the same eye measured with SD-OCT (blue dots) and histology (orange dots). IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, OLM: outer limiting membrane, RPE: retinal pigmented epithelium. SD: Standard Deviation. Scale bars: 50 µm.</p

SD-OCT imaging in other pathological models: rd8 mutation and light-challenge.

<p>(A) Typical ocular lesions of <i>rd8</i> mutation in <i>crb1</i> gene (C57BL/6NRj mice in which presence of the <i>rd8</i> mutation was confirmed by genotyping). (B–C) SD-OCT follow-up of the outer retina during a light-challenge in C57BL/6JRj mice. Control unexposed three month-old mouse has a normal appearance with 4 bands of different reflectance corresponding to the PR segments (B). Mice were then exposed to light during 4 days as described in the “methods” section and the retina was imaged by SD-OCT at day 3 (D3), 7, 14 and 21 after starting the illumination (C). The light-challenge leads to a temporary abolition of the distinction between the two bands forming the outer segment, with a peak at D7 (right panels: enlargement of the area enclosed by a white box on the left view). INL: Inner Nuclear Layer, ONL: Outer Nuclear Layer, IS: Inner Segments, OS: Outer Segments. Scale bars: 50 µm.</p

Characterization of a retinal degeneration mouse model by SD-OCT.

<p>SD-OCT images of control mice retina (A) and <i>rho−/−</i> mice retina (B) from post-natal day 21 (P21) to 180 (P180). Magnification (X2.4) of P21 and P180 control mice outer retina (C) and <i>rho−/−</i> mice (D). (E) Measures of INL thickness obtained from SD-OCT data in control and <i>rho−/−</i> mice (P21: <i>p</i> = 0.0123; P180: <i>p</i> = 0.7125). (F) Measures of ONL thickness obtained from SD-OCT data in control and <i>rho−/−</i> mice (P21 and P180: <i>p</i><0.0001). (G) Measures of ONL thickness obtained from morphometric measurements on cryostat sections in control and <i>rho</i>−/− mice (P15 and P180: p = 0.0022). Statistical significance of the difference between groups was analyzed at the initial time-point (P15 or P21) and the latest time-point (P180) studied by Student's <i>T</i>-test for E and F (n = 23 per group) and by Mann Whitney test for G (n = 6 per group). IPL: inner plexiform layer, INL: inner nuclear layer, ONL: outer nuclear layer, OLM: outer limiting membrane, RPE: retinal pigmented epithelium. SD: Standard Deviation. Scale bars: 50 µm.</p

<i>Rpe65</i> Gene Transfer Performed at 1 mo Fails to Protect Cone Photoreceptors from Degeneration

<p><i>Rpe65</i>^−/− mice were treated by subretinal injection at age 1 mo with 20 ng of lentiviral vector. Treatment at this age with the LV-RPE65 vector restored ERG response with a threshold response of 3 × 10⁻² cd s/m² for the b-wave (A), whereas no improvement was observed in the LV-GFP treated group (unpublished data). (B) Counting of PNA-positive and GNAT2-positive cells revealed that <i>Rpe65</i> gene transfer performed at age 1 mo was unable to protect the cones from degeneration. Data are presented as mean ± SEM.</p