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. 2016 Aug;27(8):580-97.
doi: 10.1089/hum.2016.085.

Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina

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Free PMC article

Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina

Shannon E Boye et al. Hum Gene Ther. .
Free PMC article

Abstract

Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. 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 are greatly enhanced.

Figures

<b>Figure 1.</b>
Figure 1.
Transduction efficiency of self-complementary AAV2-smCBA-mCherry in 661W cone photoreceptor cells. Vector was preincubated with Healon® at a 3:1 (AAV:Healon) ratio for either 5 min, 15 min, or 1 hr and then injected at a multiplicity of infection (MOI) of 2000. Controls included uninfected cells and cells infected with vector alone. mCherry expression was calculated by fluorescence-activated cell sorting (FACS) by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. Error bars represent ±1 standard deviation.
<b>Figure 2.</b>
Figure 2.
SubILM injection of AAV vector. (A) Schematic of subILM injection procedure. Arrow in (A) denotes the ILM. (B) Still photos from the injection video (1) just before and (2 and 3) during subILM injection illustrate bleb placement in nonhuman primate (NHP) subject EN-28. Arrows in (2) and (3) denote the limits of the 7.5-μl injection bleb. ILM, inner limiting membrane; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR IS, photoreceptor, inner segment; PR OS, photoreceptor, outer segment; RPE, retinal pigment epithelium.
<b>Figure 3.</b>
Figure 3.
Fluorescence fundus images and corresponding optical coherence tomography (OCT) scans in the retina of F91-108. A representative image from the earliest postinjection time point (∼3 months) reveals the location of the three subILM blebs (blue arrows) in the OD (oculus dexter, right eye), only the largest of which was injected with AAV. The extent of this subILM bleb created during surgery is denoted by the dashed yellow line. Green fluorescent protein (GFP) expression, observed at the initial smaller injection sites, may have resulted from ILM damage and subsequent leakage of vector into those regions from the vitreous. There was no apparent diminution of GFP signal in this eye until at least ∼16 months after subILM injection. OCT scans through the injection site of the largest bleb show minimal cellular displacement (red arrow), and there were no apparent structural abnormalities within the fovea. As expected, a representative image of the uninjected OS (oculus sinister, left eye) obtained during the final imaging session reveals no GFP expression or retinal disorganization. The green arrows correspond to the location in the retina through which an OCT scan was collected (OCT scans are shown on the right in each instance). Scale bars: 200 μm.
<b>Figure 4.</b>
Figure 4.
Fluorescence fundus images and corresponding OCT scans in the retina of EN-28. A representative image from ∼6 weeks postinjection shows GFP expression in a single subILM bleb in the OD. The extent of the subILM bleb created during surgery is denoted by the dashed yellow line. An OCT scan at the level indicated by the green line in the adjacent red-free image suggests that the ILM remained detached at this time point. Six months after subILM injection, an OCT scan through the injection site shows minimal cellular displacement only at the needle entry site (red arrow) and not beyond, as shown in the panel below. About 10 months after subILM injection, there was no reduction in GFP expression. However, there is evidence of a lamellar macular defect, presumably due to slight tangential traction from the ILM/epiretinal membrane. A representative fundus image and OCT scan of the intravitreally injected OS obtained at ∼10 months postinjection reveals GFP expression in the foveal ring and pit and no structural disorganization. The green arrows correspond to the location in the retina through which an OCT scan was collected (OCT scans are shown on the right in each instance). Scale bars: 200 μm.
<b>Figure 5.</b>
Figure 5.
Fluorescence fundus images and corresponding OCT scans in the retina of EV-44. A representative image from the earliest postinjection time point (∼2 weeks) reveals GFP expression in a single, small subILM bleb in the OD. The extent of the subILM bleb created during surgery is denoted by the dashed yellow line. OCT scans through this region show minimal cellular displacement, and no structural abnormalities were noted elsewhere. There was no apparent diminution of GFP signal for at least ∼15 months in the subILM-injected OD. GFP expression was not detectable in a representative fundus image of the intravitreally injected OS obtained ∼2 weeks postinjection, but was apparent ∼15 months postinjection. OCT scans showed no structural abnormalities in the OS. The green arrows correspond to the location in the retina through which an OCT scan was collected (OCT scans are shown on the right in each instance). Scale bars: 200 μm.
<b>Figure 6.</b>
Figure 6.
AAV2-CBA-mediated GFP expression in foveas of subILM-injected NHPs. Cross-sections of foveal pits of F91-108 OD (top) and EN-28 OD (bottom) were stained with an antibody raised against glial fibrillary acidic protein (GFAP; purple) and counterstained with 4′,6′-diamino-2-phenylindole (DAPI; blue). Confocal images taken at original magnifications of ×10 (left) and ×20 (right) revealed the extent of native GFP fluorescence (green) in retinal ganglion cells (RGCs), middle retina, and foveal cones of both animals. No reactive gliosis was observed in either retina as evidenced by a lack of GFAP staining. Scale bars at ×10 and ×20 represent 17 and 34 μm, respectively. ONL, outer nuclear layer; INL, inner nuclear layer; GC, ganglion cell layer.
<b>Figure 7.</b>
Figure 7.
AAV2-CBA-mediated GFP expression in retinas of subILM-injected NHPs. Cross-sections of retina from the foveal ring of EN-28 OD reveal robust native GFP expression (green) in RGCs (A). Colocalization of GFP with glutamine synthetase (red) also revealed significant transduction of Müller cells (B and C). Some cone transduction was observed in the outer retina (CE). Cross-sections from the foveal ring of F91-108 show native GFP expression in the majority of RGCs (F). Colocalization of GFP with protein kinase C α subunit (PKCα) or glutamine synthetase (red) also revealed transduction of ON bipolar cells (yellow arrows, G), and Müller glia (yellow arrow, H), respectively. Cone transduction was also observed (I). Outside the fovea, within F91-108 OD's subILM injection bleb, native GFP expression was observed in the majority of RGCs (J and K) and some Müller glia. GFP-positive RGC axons progressed through the optic nerve head of F91-108 (L). Spectralis fundus images are shown for reference (top). Confocal images are shown at original magnifications of × 20 (D, J, and L), × 40 (B, C, E–I, and K), and × 60 (A). Scale bars at × 20, × 40, and × 60 represent 34, 17, and 12 μm, respectively. ONL, outer nuclear layer; INL, inner nuclear layer; GC, ganglion cell layer; ONH, optic nerve head.
<b>Figure 8.</b>
Figure 8.
GFAP staining in subILM-injected and uninjected eyes of an NHP. Representative retinal cross-sections from F91-108 OD and OS were immunostained with an antibody raised against GFAP and counterstained with DAPI. The uninjected eye (F91-108 OS, left) had similar levels of GFAP immunoreactivity as an area outside the bleb of the subILM-injected eye (F91-108 OD, middle). There was no apparent GFAP staining immediately inside the subILM injection site of F91-108 OD (right). ONL, outer nuclear layer; INL, inner nuclear layer; GC, ganglion cell layer. Scale bar: 17 μm.
<b>Figure 9.</b>
Figure 9.
Fluorescence and dark-field images of GFP-positive axons and terminal fields. Representative sections from F91-108. Fluorescence image of GFP expression within retinal axons in the optic nerve (A). Dark-field image showing retinal projections to the core of the pretectal olivary nucleus (PON) (B). Dark-field image showing retinal projections to the superficial layers of the superior colliculus (C). Dark-field image showing retinal projections to the ipsilateral, parvocellular layers (layers 3 and 5) of the dorsal lateral geniculate nucleus (D). Dark-field image showing retinal projections to the contralateral, magnocellular layer (layer 1) of the dorsal lateral geniculate nucleus (E). Scale bars: (AC) 100 μm; (D and E) 250 μm.

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