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. 2014 Jan 15;23(2):514-23.
doi: 10.1093/hmg/ddt452. Epub 2013 Sep 18.

Mid-stage Intervention Achieves Similar Efficacy as Conventional Early-Stage Treatment Using Gene Therapy in a Pre-Clinical Model of Retinitis Pigmentosa

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Free PMC article

Mid-stage Intervention Achieves Similar Efficacy as Conventional Early-Stage Treatment Using Gene Therapy in a Pre-Clinical Model of Retinitis Pigmentosa

Katherine J Wert et al. Hum Mol Genet. .
Free PMC article

Abstract

Deficiencies in rod-specific cyclic guanosine monophosphate (cGMP) phosphodiesterase-6 (PDE6) are the third most common cause of autosomal recessive retinitis pigmentosa (RP). Previously, viral gene therapy approaches on pre-clinical models with mutations in PDE6 have demonstrated that the photoreceptor cell survival and visual function can be rescued when the gene therapy virus is delivered into the subretinal space before the onset of disease. However, no studies have currently been published that analyze rescue effects after disease onset, a time when human RP patients are diagnosed by a clinician and would receive the treatment. We utilized the AAV2/8(Y733F)-Rho-Pde6α gene therapy virus and injected it into a pre-clinical model of RP with a mutation within the alpha subunit of PDE6: Pde6α(D670G). These mice were previously shown to have long-term photoreceptor cell rescue when this gene therapy virus was delivered before the onset of disease. Now, we have determined that subretinal transduction of this rod-specific transgene at post-natal day (P) 21, when approximately half of the photoreceptor cells have undergone degeneration, is more efficient in rescuing cone than rod photoreceptor function long term. Therefore, AAV2/8(Y733F)-Rho-Pde6α is an effective gene therapy treatment that can be utilized in the clinical setting, in human patients who have lost portions of their peripheral visual field and are in the mid-stage of disease when they first present to an eye-care professional.

Figures

Figure 1.
Figure 1.
Viral spread and lack of immune response in late-degeneration treated mutant eyes. A single subretinal injection of the AAV2/8(Y733F)-Rho-Pde6α was delivered at post-natal (P) day 21, in mid-stage photoreceptor neurodegeneration as matches human patients at the time of diagnosis. Scale bar: 600 µm (A). A single subretinal injection of AAV2/8-TurboRFP into the right eye of P21 Pde6αD670G mice and visualized at P55. Red fluorescence was overlayed onto a bright-field image of the whole mount for localization of fluorescence in respect to the entire retina (B). Immunoblot analysis for antibodies against PDE6α and β-actin on retinal lysates from a C57BL/6J (B6) mouse retina. Nine micrograms of C57BL/6J (B6) retinal lysate was loaded per lane, and lanes were incubated with blood serum from a Pde6αD670G mouse 1 month after a single subretinal injection of AAV2/8(Y733F)-Rho-Pde6α (treated lane), blood serum from an untreated Pde6αD670G mouse (untreated lane), and commercial anti-PDE6α (control lane). β-Actin was used as a loading control. PDE6α antibody was not specific, and cross-reacted with the highly homologous PDE6β subunit. N = 5 mice (C).
Figure 2.
Figure 2.
Improved photoreceptor survival after AAV2/8(Y733F)-Rho-Pde6α transduction at mid-stage of disease. H&E stained retinal section of an untreated Pde6αD670G mouse retina (A), the fellow P21-treated Pde6αD670G mouse retina (B), a P5-treated Pde6αD670G mouse retina (C) and B6 control mouse (D) at 6 months of age. Scale bar: 600 µm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. Quantification of the thickness of the ONL by counting the columns of photoreceptor nuclei in the rescued half of the treated retina, the un-rescued half of the treated retina (labeled internal control) and fellow-untreated eyes from 3 to 6 months of age (E). Error bars show SEM for each time-point and the significance was calculated for the rescued half of the P21-treated retina compared with the untreated fellow eyes using paired t-test analysis. N ≥ 3 mice. **P < 0.01.
Figure 3.
Figure 3.
Rescued visual function after AAV2/8(Y733F)-Rho-Pde6α transduction. Representative scotopic maximum ERG traces for a B6 control mouse (blue), a P5-treated eye (red), a P21-treated eye (green) and an untreated eye (purple) of a Pde6αD670G mouse at 5 months of age (A). Representative scotopic dim light rod-specific ERG traces of a B6 control mouse (blue), a P5-treated eye (red), a P21-treated eye (green) and an untreated eye (purple) of a Pde6αD670G mouse at 5 months of age (B), and representative photopic single flash cone-mediated ERG traces for a B6 control mouse (blue), a P5-treated eye (red), a P21-treated eye (green) and an untreated eye (purple) of a Pde6αD670G mouse at 5 months of age (C). Maximum scotopic b-wave amplitudes in a B6 mouse, the treated Pde6αD670G eyes and fellow-untreated eyes monthly between 2 and 6 months of age (D). Maximum scotopic photoreceptor-mediated a-wave amplitudes (shown as positive values) in a B6 mouse, the treated Pde6αD670G eyes and fellow-untreated eyes monthly between 2 and 6 months of age (E). Photopic cone-specific b-wave amplitudes in a B6 mouse, the treated Pde6αD670G eyes and fellow-untreated eyes from 3 to 6 months of age (F). Error bars show SEM for each time-point and the significance was calculated for the P21-treated eyes compared with untreated fellow eyes using the ratio paired t-test analysis. N ≥ 3 mice. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4.
Figure 4.
Delay of retinal pigment epithelial atrophy after AAV2/8(Y733F)-Rho-Pde6α transduction. Representative infrared (IR) images of an untreated Pde6αD670G mutant eye at 5 (A), 6 (C), 7 (E) and 8 (G) months of age compared with the fellow-treated Pde6αD670G mutant eye at 5 (B), 6 (D), 7 (F) and 8 (H) months of age. Images for all time-points are taken from the same mouse. Representative IR image of a C57BL/6J control mouse at 6 months of age (I). Increased IR reflectance represents RPE atrophy (*). IR imaging was obtained at 790 nm absorption and 830 nm emission using a 55° lens. Images were taken of the central retina, with the optic nerve located at the center of the image and the site of the subretinal bleb along the lower left-hand quadrant.

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