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. 2011 Apr 25;52(5):2775-83.
doi: 10.1167/iovs.10-6250.

Intravitreal injection of AAV2 transduces macaque inner retina

Lu Yin  1 , Kenneth Greenberg, Jennifer J Hunter, Deniz Dalkara, Kathleen D Kolstad, Benjamin D Masella, Robert Wolfe, Meike Visel, Daniel Stone, Richard T Libby, David Diloreto Jr, David Schaffer, John Flannery, David R Williams, William H Merigan
Affiliations

Intravitreal injection of AAV2 transduces macaque inner retina

Lu Yin et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans.

Methods: In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters.

Results: AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters--human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)--AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis.

Conclusions: The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.

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Figures

Figure 1.
Figure 1.
In vivo imaging of AAV2-hCx36-GFP transduction in the macaque central retina, showing GFP expression in ganglion cells. (A) Fluorescence fundus image of GFP expression from AAV2-hCx36-GFP transduction (green image) superimposed on a fluorescein angiogram of central retina (gray image) of the same eye. Vasculature is gray in the fluorescein angiogram. The overlay shows the relationship of the GFP expression pattern, the green annulus, to the avascular zone in the fovea. (B) Enlargement of the fluorescence fundus image in (A). GFP expression is visible as an annulus around the fovea and in the bundles of labeled axons coursing from the foveal region to the optic disc. The optic disc is slightly brighter than the background and can be seen in this image, which is not from GFP expression but from the autofluorescence of the sclera. (C) AO image of a portion of retina illustrated by the white-dashed rectangle in (B). Axon bundles from transduced ganglion cell coursing toward the optic disc are visible. (D, E) AO image montages at two depths of focus of the dark-dashed rectangle in (B), covering the center and temporal side of the annulus. (D, E) Deep and more superficial focuses, respectively. Arrows: examples of transduced cell somas within the GFP-expressing annulus that are in focus in (D), but out of focus in (E); arrowheads: examples of ganglion cell somas within the annulus that are out of focus in (D), but in focus in (E). Transduced cell somas that are in sharp focus in (E) are more peripheral than those that are in sharp focus in (D) and thus are located farther up the foveal slope. In (D), a few scattered cells occupying the center of the macaque fovea (open triangle) are also visible. The difference in focus between (D) and (E) corresponds to a retinal depth of 48 μm.
Figure 2.
Figure 2.
Ex vivo (histologic) evaluation of AAV2-hCx36-GFP transduction in the macaque central retina, showing dense GFP expression within the GCL in fovea. (A) Diagram of the foveal GFP expression, illustrating the location of the images in (B–D), which are from an eye transduced with AAV2-hCx36-GFP after treatment with microplasmin. Images in (B–D) show GFP expression within the ganglion cells and their axons, not amplified with immunostaining. (B) Confocal images of the inner edge of the annulus of GFP expression in whole mount view (top) and transverse view (bottom) reconstructed from the portion of the whole mount image stack between the two dashed horizontal lines. GFP expression is visible in GCL and IPL at retinal locations away from the foveal slope. NA, 1.2. (C) Confocal image of the outer edge of the annulus of GFP expression showing dense, expressing ganglion cells and their axons. NA, 0.8. (D). Higher magnification image that partially overlaps with the portion of (C) marked by the dashed rectangle. NA, 1.2.
Figure 3.
Figure 3.
AAV2-hCx36-GFP transduction in the far peripheral retina after treatment with microplasmin. (A, B) GFP expression in ganglion cells (arrowheads) and Müller cells (arrows) from the nasal edge of the retina near the ora serrata. In the fluorescence fundus image of this eye, axon bundles from transduced ganglion cells were visible in the nerve fiber layer entering the optic disc from the nasal side (data not shown). In (B), the transverse view (right) was reconstructed from the portion of whole mount (left) image stack between the two dashed vertical lines. Transverse view shows the processes of the Müller cell (arrow) extending toward the outer retina. GFP expression was not amplified with immunostaining. NA, 1.2.
Figure 4.
Figure 4.
In vivo images of AAV2-CBA-GFP transduction in macaque central retina, showing GFP expression in both ganglion cells and Müller cells. (A) Fluorescence fundus image of GFP expression. A disc of GFP expression in Müller cells is centered on the fovea, with little GFP expression evident in the axon bundles originating from the fovea. (B) An AO image of a portion of retina illustrated by the white-dashed rectangle in (A). Axon bundles from transduced ganglion cells can be seen. The two cells labeled with arrows can be used as landmarks to visualize the relative alignment of (C) and Figure 5A. (C) Higher magnification AO image of the region marked in (B) with the white-dashed rectangle. At the edge of the disc of GFP expression, individual transduced retinal cells can be seen. Arrowhead: one of the two cells from (B). (D–G) Successive AO images of the center of the fovea (the dark-dashed rectangle in A) at four depths of focus, illustrating the variation in retinal structure. The deepest focus in (D) shows the most central processes of the Müller cells. The most superficial focus in (G) shows the foveal Müller cell processes out of focus and the more superficial ganglion cells in focus. (E, F) The intermediate focus steps, where the processes of Müller cells gradually extend from the outer retina toward the inner retina. A few Müller cell somas are labeled with arrows in (E, F). The focus step of 0.15 D corresponds to a retinal depth of 36 μm. Scale bar: (E–G) same as in (D).
Figure 5.
Figure 5.
Ex vivo (histologic) evaluation of AAV2-CBA-GFP transduction in the macaque central retina. (A) Confocal image of the retinal region illustrated in Figure 4B by the red-dashed rectangle. The two retinal cells marked by arrowheads are the same cells marked by arrowheads in Figure 4B. (B) Confocal images of a transverse section through the center of the fovea, illustrating GFP expression in ganglion cell somas in the GCL surrounding the fovea and Müller cell processes extending from the outer to the inner retina. The images from left to right overlap, but differ slightly in focal plane. The sclerad end of the processes of GFP-expressing Müller cells were located close to the foveal center (deep), and gradually course away from the fovea center toward their vitread end in the ILM above the GCL. The thickness of the sections was 70 μm. Arrows: Müller cell somas. (A, B) GFP expression was amplified with immunostaining; NA, 0.8.
Figure 6.
Figure 6.
Ex vivo (histologic) images of AAV2-CBA-GFP transduced peripheral Müller and ganglion cells. (A) Montage of confocal images of a strip of peripheral retina extending from the ora serrata ∼5.2 mm (∼23°) toward the central retina, showing GFP expression in both scattered cells and clusters of cells. The density of transduced retinal cells declined toward the central retina (arrow). NA, 0.16. (B–D) Through-focus at higher magnification of the area marked by the rectangle in (A), showing GFP-expressing ganglion cells and Müller cells. Most transduced cells are Müller cells, shown as the dense GFP-expressing processes at the retinal surface (B) and the sclerad end of the processes (D; ∼43 μm deeper than B) in the outer retina. (C, arrow) A ganglion cell, ∼11 μm deeper than (B). In (A–D) GFP expression was amplified with immunostaining; NA, 1.2.
Figure 7.
Figure 7.
Fundus images illustrating three shorter promoters used with AAV2 that resulted in little or no visible GFP expression in normal retinas (A, C, E), but visible GFP expression in eyes with altered retinas (B, D, F). (A, B) Neuronal promoter, hSYN, produced no visible GFP expression after 4.5 months (A), but when injected into an eye with ganglion cell loss, (B) an annulus of GFP expression was seen in less than 2 months (data not shown; instead a later fundus image after 11 month is shown for better image quality). The transduction pattern in (B) is similar to that shown in Figure 1A, but the intensity of GFP expression is lower. (C, D) Shortened hCx36sh promoter resulted in no visible GFP expression after 4.5 months (C; for comparison, <2 months was typically needed for transduction by the full-length hCx36 promoter). However, when the promoter was injected into an eye with ganglion cell loss (D), a faint annulus of GFP expression around the fovea center was visible less than 3.5 months when the first fundus image was acquired (data not shown; instead a later fundus image after 4.5 months was shown for better image quality.) The transduction pattern in (D) was similar that shown in Figure 1A, but the intensity of GFP expression was lower. (E, F) Shortened CBA promoter (smCBA) resulted in no GFP expression after 15 months when injected into normal retina (E; for comparison, <10 months was needed for transduction by full-length CBA promoter). However, when injected into an eye that received enzymatic vitreolysis with microplasmin (F), GFP expression in fovea was visible in less than 4.5 months, when the first fundus image was acquired (data not shown; instead a later fundus image after 10 months is shown for better image quality). The transduction pattern in (F) was similar to that shown in Figure 4A.
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