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. 2019;1950:249-262.
doi: 10.1007/978-1-4939-9139-6_14.

SubILM Injection of AAV for Gene Delivery to the Retina

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Free PMC article

SubILM Injection of AAV for Gene Delivery to the Retina

Paul D Gamlin et al. Methods Mol Biol. .
Free PMC article

Abstract

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retinal structure and visual acuity in some patients. Due to its technical difficulty and potential risks, alternatives to SR injection have been explored in primates. Intravitreally (Ivt) delivered AAV transduces inner retina and foveal cones, but with low efficiency. Novel AAV capsid variants identified via rational design or directed evolution have offered only incremental improvements, and have failed to promote pan-inner retinal transduction or significant outer retinal transduction beyond the fovea. Problems with retinal transduction by Ivt-delivered AAV include dilution in the vitreous, potential antibody-mediated neutralization of capsid in this nonimmune privileged space, and the presence of the inner limiting membrane (ILM), a basement membrane separating the vitreous from the neural retina. We have developed an alternative "subILM" injection method that overcomes all three hurdles. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. We have shown that subILM injection promotes more efficient retinal transduction by AAV than Ivt injection, and results in uniform and extensive transduction of retinal ganglion cells (RGCs) beneath the subILM bleb. We have also demonstrated transduction of Muller glia, ON bipolar cells, and photoreceptors by subILM injection. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes is greatly enhanced. Here we describe in detail methods for vector preparation, vector dilution, and subILM injection as performed in macaque (Macaca sp.).

Keywords: AAV; Bipolar cells; Gene delivery; Gene replacement; Inner limiting membrane; Novel surgical technique; Photoreceptors; Retinal ganglion cells.

Figures

Fig. 1
Fig. 1
SubILM injection of AAV and Healon. A schematic of an intact retina (left) is magnified (right) to reveal the location of the subILM injection bleb. A needle is placed posterior to the ILM and anterior to NFL and GCL. Approximately 10 μL of a 1:1 solution of AAV:healon is injected into this space. RPE retinal pigment epithelium, PR OS photoreceptor outer segments, PR IS photoreceptor inner segments, ONL outer nuclear layer, OPL outer plexiform layer, IN L inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, NFL nerve fiber layer, ILM inner limiting membrane
Fig. 2
Fig. 2
Fluorescence fundus image of AAV-mediated GFP expression in NHP subject EN-28 taken 4 months postinjection. Healon and AAV (1.7 × 1012 vector genomes per mL) were subILM-injected at a ratio of 1:1. Total bleb volume was 7.5 μL and contained a total of 6.2 × 109 vector genomes. Scale bars = 400 μm
Fig. 3
Fig. 3
AAV2-CBA-mediated GFP expression in subjects EN-28 and F91–108. Raw GFP (green), DAPI (blue), and Glial fibrillary acidic protein (purple) are shown in all panels. Glutamine synthetase (red) is shown in panel D. The vast majority of retinal ganglion cells within the subILM injection blebs are GFP positive (a, b). Transduction of photoreceptors (white arrows in a, b, d) and Muller glia (c, d) is also observed. IS/OS inner segments/outer segments, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer. Scale bars = 70 μm (a), 35 μm (b), 17 μm (c, d)

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