Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse

Abstract

X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11-15 weeks after the application.

Figure 1

Retinal enhanced green fluorescent protein (EGFP) expression after intravitreal injection of adeno-associated virus type 8 (AAV8) CMV EGFP vector. A dose of 1.5 × 10¹¹ vg of AAV8 CMV EGFP vector was administered to wild-type (a, b) and retinoschisin knockout (Rs1-KO) mice (c, d) at 8 weeks of age, and older Rs1-KO mice (e, f) at 7 months of age. The mice were killed at 8 weeks post injection (PI) and ocular sections were prepared. EGFP expression (green) is shown in the retinal sections of the mice mentioned above (a–f), and in the optic nerve (g), retinal nerve fiber layer (RNFL) (h), ciliary body (i) and cornea (j) of Rs1-KO mice. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Calibration bar, 100 µm unless otherwise marked.

Figure 2

Schematic illustration of the adeno-associated virus (AAV) 8-hRSp4 vector genome. A 3429 bp human retinoschisin promoter drives the expression of a human retinoschisin cDNA containing a 150 bp truncated retinoschisin first intron. The promoter fragment has a 626 bp deletion of genomic repetitive sequence, which corresponds to base pairs −1286 to −1911 relative to the translational start codon. A 218-bp fragment encoding the human β-globin 3′ untranslated region and polyadenylation site mediates polyadenylation. The inverted terminal repeats (ITRs) are derived from AAV2. CpG indicates a CpG island.

Figure 3

Retinoschisin expression after vitreal administration of adeno-associated virus type 8 (AAV8)-hRSp4 vector to retinoschisin knockout (Rs1-KO) mice. Rs1-KO mice of 9-week-old received intravitreal injections of 7.5 × 10¹⁰ vg of AAV8-hRSp4 vector. Animals were killed at 8 and 11 weeks post injection and retinal sections were prepared. Fluorescence images were taken under low (×200) and high (×400) magnification. Immunolabeling of retinoschisin (RS) was visualized with Alexa-568-conjugated goat-anti-rabbit IgG (red). The nuclei were stained blue with 4′,6-diaminidino-2-phenylindole (DAPI) in some sections. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 4

Retinoschisin expression in wild-type (WT) mice, retinoschisin knockout (Rs1-KO) mice, and Rs1-KO mice treated with adeno-associated virus (AAV) 2 CMV mRS and AAV8-hRSp4 vectors. Vector treated mice received intravitreal injections of 2.3 × 10¹⁰ vg of AAV2 CMV mRS or 7.5 × 10¹⁰ vg of AAV8-hRSp4 at 9 weeks of age. Vector treated animals were killed at 8 and 11 weeks post injection (PI) (panels c–f). The untreated animals were killed at 20 weeks of age (panels a and b). Immunolabeling of retinoschisin (RS) was visualized with Alexa-568-conjugated goat-anti-rabbit IgG (red). Some sections were also stained with 4′,6-diaminidino-2-phenylindole (DAPI) to indicate nuclei (blue). IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; Wt, wild type. Calibration bar, 40 µm.

Figure 5

Gene expression patterns in extra-retinal tissues intravitreal injection of adeno-associated virus (AAV) 2 CMV mRS and AAV8-hRSp4 vectors. Retinoschisin knockout mice of 9-week-old received intravitreal injections of AAV2 CMV mRS, and AAV8-hRSp4 vectors at doses of 2.3 × 10¹⁰ and 7.5 × 10¹⁰ vg per eye, respectively. AAV2 CMV mRS, and AAV8-hRSp4 vector-treated mice were killed at 11 weeks post injection. Immunolabeling of retinoschisin was visualized with Alexa-568-conjugated goat-anti-rabbit IgG (red). Some sections were also stained with 4′,6-diaminidino-2-phenylindole (DAPI) to indicate nuclei (blue). AAV2 CMV mRS-treated mice showed expression in the optic nerve and the retinal nerve fiber layer (RFNL), but not in the ciliary body or peripheral cornea (a). AAV8-hRSp4 vector-treated mice did not show retinoschisin expression in the optic nerve, RNFL or uveal tissue (b). Retinoschisin expression in RNFL was marked with double asterisk.

Figure 6

Box and whiskers plot of b/a-wave ratios from Table 1 showing minimum and maximum, 25th and 75th percentile and median. (a) (Experiment 1) Retinoschisin knockout (Rs1-KO) mice were treated in both eyes (n = 8 eyes) with adeno-associated virus (AAV) 8-hRSp4 vector. Injections were performed at 6–7 weeks of age, and electroretinograms (ERGs) were recorded 11 weeks later. Age-matched untreated Rs1-KO mice were used as controls (n = 6 eyes), P = 0.04. Open circle is an outlier (1.5–3.0 times the interquartile range), which was included in the P-value calculation. (b) (Experiment 2) Rs1-KO mice were treated in one eye with AAV8-hRSp4 vector. Injections were done at 7–8 weeks of age, and ERGs were recorded 14.7–15.7 weeks later (P = 0.012, n = 5). The area between the dashed lines indicates the b/a-ratio range from a group of 39 wild-type animals.