Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness

Abstract

Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.

Figure 1.

Glutamate cascade proteins expressed in dendritic tips of ON BCs. Glutamate released from photoreceptor terminals in the dark binds the mGluR6 metabotropic glutamate receptor and activates the alpha subunit of a heterotrimeric G-protein, Gα_o. Dissociation of G-protein subunits culminates in closure of the non-selective cation channel, TRPM1. Gα_o is inactivated by the β subunit (β5) and by a GTPase activating the regulator of G-protein signaling complex. A leucine-rich repeat protein, NYX, is required for the correct localization of TRPM1, whereas GPR179, another G-protein receptor, mediates channel sensitivity. LRIT3 interacts with both NYX and TRPM1.

Figure 2.

AAV2(quadY-F+T-V)-Ple155-mediated GFP expression (green) is restricted to ON BCs as evidenced by co-staining with either rod bipolar marker PKCα (A and C) or ON BC marker PCP2 (B) in red. Only minimal transduction was achieved via subretinal injection (A), but robust and targeted ON BC transduction was observed following P30 intravitreal injection in both WT (B) and Nyx^nob mice (C). Panretinal transduction of ON BCs was observed following intravitreal injection in both strains (WT shown in D). SR = subretinal, Ivt = intravitreal, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bars = 34 μm (A–C) and 200 μm (D).

Figure 3.

Intravitreal AAV2(quadY-F+T-V)-Ple155-YFP_Nyx transduction (green, GFP antibody) is restricted to ON BCs (red, PKCα antibody). Widespread ON BC transduction is observed in whole eyecups of P2-injected Nyx^nob mice (A). Intraocular injection at P2 promoted robust transduction of ON BCs in both strains (Nyx^nob mouse shown) (B and C). High magnification (white box) reveals YFP_NYX expression in cell bodies and dendritic tips of ON BCs (C). Minimal YFP_NYX expression was observed following intravitreal injection in P30 WT or Nyx^nob mice (D and E). ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bars = 17 μm (A–D) and 200 μm (E).

Figure 4.

Electroretinographic analysis of AAV2(quadY-F+T-V)-Ple155-YFP_Nyx-treated, AAV2(quadY-F+T-V)-Ple155-GFP-treated and untreated Nyx^nob mice. Representative dark-adapted (A, left) and light-adapted (A, right) ERGs obtained from the two eyes of an individual mouse. Red traces were recorded from the untreated eye, while black traces were recorded from eye in which AAV-YFP_Nyx was injected. Note the presence of a positive signal (arrowheads) in the treated responses, which was not present in the responses obtained from the untreated eye. Values to the right of each pair of waveforms indicate strobe flash luminance in log cd sec/m². The average positive response measured at 100 ms after stimulus presentation from AAV-YFP_Nyx (black) and untreated (red) eyes to a −2.4 log cd sec/m² stimulus (B). The left and right pairs of bars compare responses from eyes treated with AAV-YFP_Nyx and AAV-GFP, respectively. Error bars report standard error of the mean. Div = division.

Figure 5.

TRPM1 localization is restored to membranes of ON BC dendritic tips in treated Nyx^nob mice. Top-down view of treated retina showing TRPM1 (red) and YFP_NYX (green, GFP antibody) in cell bodies (A, top row) and dendritic tips (A, middle row). Dendritic tip colocalization is also shown in retinal cross section (A, bottom row) and a representative ON BC at high magnification (B). OPL = outer plexiform layer, INL = inner nuclear layer. Scale bars = 10 μm (A) and 5 μm (B).

Figure 6.

ON BC signaling is restored following AAV treatment in Nyx^nob mice. A representative rod BC expressing AAV-mediated YFP_NYX and properly localized TRPM1 was targeted for whole-cell patch-clamp recording and filled with sulforhodamine B (A). Capsaicin (10 μM), a TRPM1 agonist, was puffed onto the rod BC dendrites to gate the opening of the TRPM1 channel. V_hold = 50 mV. Representative current recordings from WT rod BC, AAV-treated and untreated rod BCs from Nyx^nob mice reveal full restoration of ON BC signaling following treatment (B). Capsaicin was puffed onto rod BC dendrites for 200 ms and 1 s durations. Current responses were recorded from each cell and then averaged (C).

Figure 7.

YFP_NYX mRNA is detectable in both early (P2)- and late (P30)-treated Nyx^nob retinal extracts through qRT-PCR. Standard deviations were calculated from three replicate reactions performed for each sample.