Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye

Abstract

Purpose: In an earlier study we found normal adeno-associated viral vector type 2 (AAV2)-mediated GFP expression after intravitreal injection to one eye of normal C57BL/6J mice. However, GFP expression was very poor in the partner eye of the same mouse if this eye received an intravitreal injection of the same vector one month after the initial intravitreal injection. We also found both injections worked well if they were subretinal. In this study, we tested whether the efficiency of subretinal AAV vector transduction is altered by a previous intravitreal injection in the partner eye and more importantly whether therapeutic efficiency is altered in the rd12 mouse (with a recessive RPE65 mutation) after the same injection series.

Methods: One microl of scAAV5-smCBA-GFP (1 x 10(13) genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old C57BL/6J mice and 1 microl of scAAV5-smCBA-hRPE65 (1 x 10(13) genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old rd12 mice Four weeks later, the same vectors were subretinally injected into the left eyes of the same C57BL/6J and rd12 mice. Left eyes of another cohort of eight-week-old rd12 mice received a single subretinal injection of the same scAAV5-smCBA-hRPE65 vector as the positive control. Dark-adapted electroretinograms (ERGs) were recorded five months after the subretinal injections. AAV-mediated GFP expression in C57BL/6J mice and RPE65 expression and ERG restoration in rd12 mice were evaluated five months after the second subretinal injection. Frozen section analysis was performed for GFP fluorescence in C57BL/6J mice and immunostaining for RPE65 in rd12 eyes.

Results: In rd12 mice, dark-adapted ERGs were minimal following the first intravitreal injection of scAAV5-smCBA-RPE65. Following subsequent subretinal injection in the partner eye, dramatic ERG restoration was recorded in that eye. In fact, ERG b-wave amplitudes were statistically similar to those from the eyes that received the initial subretinal injection at a similar age. In C57BL/6J mice, GFP positive cells were detected in eyes following the first intravitreal injection around the injection site. Strong GFP expression in both the retinal pigment epithelium (RPE) and photoreceptor (PR) cells was detected in the partner eyes following the subsequent subretinal injection. Immunostaining of retinal sections with anti-RPE65 antibody showed strong RPE65 expression mainly in the RPE cells of subretinally injected eyes but not in the intravitreally injected eyes except minimally around the injection site.

Conclusions: These results show that an initial intravitreal injection of AAV vectors to one eye of a mouse does not influence AAV-mediated gene expression or related therapeutic effects in the other eye when vectors are administered to the subretinal space. This suggests that the subretinal space possesses a unique immune privilege relative to the vitreous cavity.

Figure 1

AAV5-mediated GFP expression both in retinal whole mounts and frozen sections following intravitreal and subretinal injection of scAAV5-smCBA-GFP. Higher transduction efficiency is seen following subretinal injection compared to intravitreal injection. A: Fluorescence image of a retinal whole-mount six months following intravitreal injection. B: Fluorescence image of a frozen section six months following intravitreal injection. C: Fluorescence image of a retinal whole-mount five months after subretinal injection (the partner eye of this mouse was pretreated with the same vector by intravitreal injection one month earlier). D: Fluorescence image of a frozen section as in (C). Abbreviations: RPE represents retinal pigment epithelium; IS represents inner segments of photoreceptor cell; ONL represents outer nuclear layer; INL represents inner nuclear layer; GCL represents ganglion cell layer.

Figure 2

RPE65 immunoreactivity after intravitreal and subretinal injections of scAAV5-smCBA-hRPE65 vector. Little RPE65 expression was detected in an rd12 eye six months after the intravitreal injection (B), which was further supported by a higher magnification image (D). RPE65 expression was detected in the RPE cells of the contralateral eye from the same rd12 mouse five months after subretinal injection (A), which was further supported by a higher magnification image (C). Abbreviations: RPE represents retinal pigment epithelium; OS represents outer segments of photoreceptor cell; IS represents inner segments of photoreceptor cell; ONL represents outer nuclear layer; INL represents inner nuclear layer; GCL represents retinal ganglion cell layer. The asterisk shows retinal detachment.

Figure 3

Dark-adapted (rod-derived) ERG analysis showing retinal function of seven-month-old normal and rd12 mice following either intravitreal or subretinal injection, or no injection. Restored ERGs are independent of whether the partner eye was pretreated with the same vector or not. Dark-adapted photoresponses are shown at different input flash intensities from an uninjected normal C57BL/6 (A) and an uninjected rd12 eye (B). Panel C shows the ERGs from the left eye of an rd12 mouse five months after subretinal injection at eight weeks of age, while the right eye of the same mouse had received intravitreal vector four weeks before the subretinal injection (E). Panel D shows ERGs from an rd12 eye five months following only a subretinal injection at eight weeks of age with no prior treatment of the partner eye.

Figure 4

ERG signals following different injections. Dark-adapted ERG b-wave amplitudes under different intensities show dramatic ERG restoration in rd12 mice 5 months following subretinal injection. Statistical analysis demonstrates that functional restoration following subretinal injection is independent of whether the partner eye was pretreated with the same vector or not. Each of the five groups had three mice. The yellow curve represents the uninjected, normal C57BL/6J eyes at seven months of age. The blue curve represents the rd12 eyes at seven months of age following an initial subretinal injection of scAAV5-smCBA-hRPE65 when they were eight weeks old. The brown curve represents the right eyes of rd12 mice six months following the intravitreal injection that occurred when they were four weeks old. The green curve represents the left eyes of rd12 mice five months following the subretinal injection they received when they were eight weeks old (the right eyes of these mice received the intravitreal vector four weeks before their subretinal injections). The pink curve represents the untreated rd12 eyes at seven months of age. Bars: mean±SD.