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

<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response of 3 × 10⁻² cd s/m² for the b-wave (A), whereas no improvement was observed in the LV-GFP treated group (unpublished data).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

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

High Dose of LV-RPE65 Vector Restores a “Near Normal” ERG Response in <i>Rpe65</i>^−/− Mice and Rescues the Activity of Cone Photoreceptors in <i>Rpe65</i>^−/−/<i>Gnat1a</i>^−/− Mice

<div><p>(A) Scotopic ERG recorded at age 1 mo of a <i>Rpe65</i>^−/− mouse treated at P5 with 180 ng of LV-RPE65 (left tracings) and of a <i>Rpe65</i>^+/+ untreated mouse (WT, right tracings). At this dose, LV-RPE65 treatment restored an ERG response similar to a wild-type response. For <i>Rpe65</i>^−/− eyes showing the best restoration (<i>n</i> = 3), thresholds of both the a-wave and the b-wave reached normal levels with, respectively, 10−¹ and 10−³ cd s/m².</p> <p>(B and C) At P5 <i>Rpe65</i>^−/−/<i>Gnat1a</i>^−/− mice received a bilateral intravitreal injection of the vector (LV-RPE65 or LV-GFP) at a dose of 180 ng of p24 capsid protein. 1 mo later, the ERG was recorded in scotopic (B) and in photopic conditions (C). Vertical bars indicate occurrence of the stimuli; x-axis: 40 ms/div; y-axis: 100 μV/div (left tracings), except right tracings in (A) (200 μV/div).</p></div

PNA and GNAT2 Labelling Increase after LV-RPE65 Treatment at P5 of <i>Rpe65</i>^−/− Mice

<div><p>(A and B) PNA linked to TRITC labels cone outer segments in red in a LV-RPE65-treated eye (A). (B) Merged picture representing RPE65 (green), PNA labelling (red), and cell nuclei (blue) in the same section used in (A).</p> <p>(C) PNA labelling was quantified by counting the number of PNA-positive outer segments in the sagittal section that bisected the optic nerve for each eye of LV-RPE65 (<i>n</i> = 9) or LV-GFP (<i>n</i> = 7) treated groups as well as of <i>Rpe65</i>^−/− (KO; <i>n</i> = 4) and wild-type control groups (<i>n</i> = 4). Values represent percentage of wild type (set to 100%). A significant increase of PNA labelling was seen for LV-RPE65 treated group compared to the LV-GFP (<i>n</i> = 7, <i>p</i> = 0.007) or untreated (<i>n</i> = 4, <i>p</i> = 0.018) groups (*<i>p</i> < 0.05). Data are presented as a mean percentage of the wild-type value ± SEM.</p> <p>(D) GNAT2 (revealed in green by a secondary antibody linked to FITC) in a wild-type retina.</p> <p>(E) The GNAT2 staining is extremely weak in <i>Rpe65</i>^−/− mice at age 4 mo. *Example of remaining outer segment staining counted in the <i>Rpe65</i>^−/− and LV-GFP treated groups.</p> <p>(F) Quantification of GNAT2 labelling was performed (as described for PNA labelling) and showed a significant increase of GNAT2 expression in the LV-RPE65 group (<i>n</i> = 6) compared to the LV-GFP (<i>n</i> = 8, <i>p</i> = 0.017) or untreated (<i>n</i> = 4, <i>p</i> = 0.03) groups (*<i>p</i> < 0.05). The discrepancy in cone loss between PNA and GNAT2 markers in <i>Rpe65</i>^−/− (compare control groups in C and F) is most probably due to the persistence of the cone interphotoreceptor matrix sheath, which can still be present after other cone markers have disappeared [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030347#pmed-0030347-b042" target="_blank">42</a>]. Data are presented as a mean percentage of the wild-type value ± SEM.</p> <p>(G and H) A GNAT2 pattern similar to that observed in wild-type mice is recovered after LV-RPE65 treatment in <i>Rpe65</i>^−/− mice (G). Double labelling shows GNAT2 stained in red, RPE65 in green (H). Example of outer segment positive for GNAT2 is pointed by an arrowhead.</p> <p>(I) LV-GFP treatment does not improve GNAT2 expression in <i>Rpe65</i>^−/−. Example of weak residual labelling for GNAT2 in a control eye treated with LV-GFP (arrow).</p> <p>(J) After LV-RPE65 treatment, a transitional area with spared GNAT2-positive cones but devoid of RPE65 expression was observed (dashed line).</p> <p>(K and L) In (K) the diagram depicts a transitional area in a ring (red) surrounding the transduced area (green). The radius of the transduced area is indicated by x; the width of the transitional area is indicated by y. The relation of x to y is graphed in (L); the radius of the transduced area is linearly correlated with the transitional area.</p> <p>Scale bar in (I): 50 μm in A, B, E, and G–I; 100 μm in D and J. KO, <i>Rpe65</i>^−/− mice; LV-GFP, LV-GFP-treated mice; LV-RPE65, LV-RPE65-treated mice; WT, <i>Rpe65</i>⁺^/⁺ mice.</p></div

Range of visual function in GUCY2D^E837D/R838S transgenic pigs.

<p>(A) Traces of photopic electroretinogram recordings (ERG) at 11 weeks are shown for one representative control and several transgenic animals. (A, first column) single flash at 3 cds/m², (A, second column) single flash at 10 cds/m2, (A, third column) flickers at 3 cds/m²,30 Hz. (B) b-wave amplitudes for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (C) a-wave amplitudes obtained for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (D) Representation of the time needed to complete the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. (E) Representation of the errors (missing or striking into an obstacle), alternative prospections (the number of times individuals investigated an obstacle by sniffing or licking) and resulting scores from the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. Horizontal bars in B,C represent the mean of the different groups with the SEM; *: p<0.05;***: p<0.001; errors in E: miss or strike into an obstacles; alternative prospection in E: sniff or lick the obstacles; score in E: sum of errors and alternative prospections.</p

Case reports: pig 917.

<p>Grouped data for the transgenic pig 917 (3 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 917 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–P) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), GFAP (H,I) and S-opsin (I,J,O,P) in the central region of the retina of pig 917 (E–J) and a non-transgenic control (K–P). Arrows in E, G, K and M show examples of M-opsin positive outersegment, arrowhead in G shows displaced nuclei, arrowhead in J shows a displaced nucleus in a S-opsin positive cell. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–J represents 50 µm.</p

Case reports: pig 920.

<p>Grouped data collected for the transgenic pig 920 (4 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 920 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–L) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), and GFAP (H,I) in the central region of the retina of pig 920 (E–H) and a non-transgenic control (I–L). Arrows in E, G, I and K show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–L represents 50 µm.</p

Expression of GUCY2D^E837D/R838S transcript in transgenic pigs.

<p>Assessment of transgene expression by RT-PCR. Transgenic pigs: 904, 907, 908, 914, 915, 917, 918, 920, control non-transgenic pig (929). +: with reverse transcription; −: without reverse transcription; hGUCY2Dmut: GUCY2D^E837D/R838S PCR fragment; pig GUCY2D: pig GUCY2D PCR fragment; GAPDH: pig GAPDH PCR fragment.</p

Case report: pig 908.

<p>Grouped data for the transgenic pig 908 (2 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 908 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). For technical reasons, the time to reach the end of the obstacle course has not been measured at 52 weeks. (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–G) Immunolabeling for M-opsin (E), PNA (F) and merged picture (G) in the central region of the retina of pig 908. Arrows in E and G show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–G represents 50 µm.</p