Highly sensitive visual restoration and protection via ectopic expression of chimeric rhodopsin in mice

Abstract

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops into Gloeobacter rhodopsin, thereby generating Gloeobacter and human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.

Keywords: Behavioral neuroscience; Molecular neuroscience; Neurology.

Graphical abstract

Figure 1

Ectopic GHCR expression restores light responses in the rd1 mouse retina (A) DNA expression cassette schematic. The GHCR coding sequence is driven by the CAGGS promoter, flanked by inverted terminal repeats (ITR), and stabilized by a polyadenylation signal sequence (pA) and a woodchuck hepatitis posttranscriptional regulatory element (WPRE). (B, C, and E) Raster plots and peri-stimulus time histograms for light stimulation of control (AAV-DJ-CAGGS-EGFP) (B), GHCR-treated (AAV-DJ-CAGGS-GHCR) (C), and coGHCR-treated (AAV-DJ-CAGGS-coGHCR) mice (E). Responses to exposure to a white LED with varying light intensity for 1.0 s. Gray shading around the averaged traces represents the standard error of the mean (SEM). (D) Confocal image of a transverse rd1 mouse retina section 2 months after AAV-DJ-CAGGS-coGHCR intravitreal injection. Green, FLAG tag antibody signal (vector); red, PKCα signal (bipolar cells); blue, 4′,6-diamidino-2-phenylindolenuclear (DAPI) counterstaining. Scale bar, 50 μm. (F) Quantitation of the firing rates of RGCs transduced with GHCR or coGHCR at the indicated light intensity. (G) Histogram showing the number of RGCs that responded to light per unit area (2.6 mm²) of the retinas of GHCR- or coGHCR-treated mice (n = 3 each). (H) Changes in cAMP consumption in response to Gi/o-coupled G-protein-coupled receptor activation in HEK293T cells transfected with GHCR and coGHCR (n = 3 each). (I) Spectral sensitivity induced by coGHCR (n = 23 cells each). Error bars represent the SEM. Data were analyzed with Student’s two-tailed t-test in (F and G) and one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test in (H); ∗ represents p ≤ 0.05, ∗∗ represents p ≤ 0.01, and ∗∗∗ represents p ≤ 0.001. GHCR, Gloeobacter and human chimeric rhodopsin; coGHCR, codon-optimized Gloeobacter and human chimeric rhodopsin; GCL, ganglion cell layer; INL, inner nuclear layer.

Figure 2

coGHCR restored vision in rd1 mice through the primary visual cortex (A) Schematic view of the VEP recording strategy. (B) Representative VEP traces from GHCR-treated, coGHCR-treated, and control mice. (C) The average amplitude of the VEPs in the control (AAV-DJ-CAGGS-EGFP, n = 6), GHCR-treated (AAV-DJ-CAGGS-GHCR, n = 8), and coGHCR-treated (AAV-DJ-CAGGS-coGHCR, n = 6) mice. The stimulus was a white LED flash (3 cd s/m²). Signals were low-pass filtered at 300 Hz and averaged over 60 trials. Error bars represent the SEM. Data were analyzed with one-way ANOVA and Tukey’s multiple comparison test; ∗ represents p ≤ 0.05. V1, visual cortex; LGN, lateral geniculate nucleus; GHCR, Gloeobacter and human chimeric rhodopsin; coGHCR, codon-optimized Gloeobacter and human chimeric rhodopsin.

Figure 3

coGHCR-treated mouse behavior indicated vision restoration (A) LDT testing schematic. Mice were tested in a 30 × 45 × 30-cm box with equally sized bright and dark chambers connected by a 5 × 5-cm opening, across which the mice could move freely. (B and C) Percentage of time spent in the bright area (total, 10 min) by wild type (n = 4), and control (AAV-DJ-CAGGS-EGFP) (n = 7 in (B) and n = 4 in (C)) and coGHCR-treated (AAV-DJ-CAGGS-coGHCR) rd1 mice (n = 6). LDT test at 3 months (B) and 2 years (C) after treatment, 10 lux illumination. (D and E) The percentage of time spent in the bright area (total, 10 min) by wild type (n = 6), and control (AAV-6-CAGGS-EGFP) (n = 8), coGHCR-treated (AAV-6-CAGGS-coGHCR) (n = 6), ChrimsonR-treated (AAV-6-CAGGS-ChrimsonR) (n = 6), and human rhodopsin-treated (AAV-6-CAGGS-human-rhodopsin) rd1 mice (n = 6). LDT test with 3,000 lux (D) and 10 lux (E) illumination. (F) VRT setup. Time spent in areas showing a video of mice fighting (object half, blue) or an empty cage (control half, red) was measured. (G) Distribution of time spent in the object half by wild type (n = 14), and control (no treatment) (n = 23), AAV-2-coGHCR-treated (AAV-2-CAGGS-coGHCR) (n = 30), AAV-DJ-coGHCR-treated (AAV-DJ-CAGGS- coGHCR) (n = 33), and AAV-DJ-C1V1-treated (AAV-DJ-CAGGS-C1V1) rd1 mice (n = 20). LDT test with 10 lux (D) and 3,000 lux (E) illumination. Black line, average value. Error bars represent the SEM. Data were analyzed with one-way ANOVA and Tukey’s multiple comparison test; ∗ represents p ≤ 0.05. coGHCR, codon-optimized Gloeobacter and human chimeric rhodopsin.

Figure 4

Ectopic coGHCR expression protects against photoreceptor degeneration (A) Confocal image of a transverse section through the P23H retina 2 months after AAV-DJ-CAGGS-coGHCR subretinal injection. Green, FLAG tag fused to the C-terminus of coGHCR; blue, DAPI nuclear counterstaining. Scale bar, 100 μm. (B) OCT retinal image sections from coGHCR-treated and control (AAV-DJ-CAGGS-EGFP subretinally injected) mice at PND 30. The white arrow indicates the measured ORT (from ONL to cone outer segment). Scale bar, 20 μm. (C) Histogram of the measured ORT of the coGHCR-treated (n = 13) and control mice (n = 10) at PND 30. (D and E) Representative ERG waveforms (rod response, mixed response, and cone response) of coGHCR-treated (n = 14) and control mice (n = 9) (D). Histograms of the average ERG amplitudes from panel d at PND 30 (E). Error bars represent SEM. Data were analyzed with the unpaired t-test; ∗∗∗ represents p ≤ 0.001. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; GHCR, coGHCR, codon-optimized Gloeobacter and human chimeric rhodopsin; ORT, outer retinal thickness.

Figure 5

coGHCR treatment suppressed retinal apoptosis and ER stress (A and B) TUNEL-stained transverse sections (A) and enlarged images of the white squares (B) of coGHCR-treated and control (AAV-DJ-CAGGS-EGFP subretinally injected) mouse retinas at PND 31. Red, TUNEL-positive cells; blue, DAPI nuclear counterstaining. Scale bar, 1,000 μm in (a) and 100 μm in (B). (C) Histogram of the number of TUNEL-positive cells in the ONLs of coGHCR-treated (n = 3) and control mice (n = 3) at PND 31. (D–G) (D) TEM images of transverse sections from coGHCR-treated and control mice at PND 31, showing the outer retinal layer (D), the outer segment at low magnification (E) and high magnification (F), and the inner segment (G). The arrowhead indicates swollen ER. Scale bar, 20 μm in (D), 5 μm in (E), 1 μm in (F), and 500 nm in (G). (H) Chromatograms of retinal in mouse retina analyzed by HPLC. 15 h dark adapted mice were exposed to light of 1000 lux for 10 min and each retina was processed and retinal oximes extracted under dim red light. Peak identification was determined using retinal standard reagents as follows: 1, syn-11-cis-retinal oxime; 2, syn-all-trans-retinal oxime; 3, anti-11-cis-retinal oxime; 4, anti-all-trans-retinal oxime. (I and J) Histogram quantifying the amount of retinal oximes from coGHCR-treated (n = 9) and control mice (n = 9) obtained from HPLC. Error bars represent SEM. Data were analyzed with the unpaired t-test; ∗ represents p ≤ 0.05, ∗∗ represents p ≤ 0.01. coGHCR, codon-optimized Gloeobacter and human chimeric rhodopsin; RPE, retinal pigment epithelium; ONL, outer nuclear layer.