Optogenetic gene therapy holds promise to restore high-quality vision in blind patients and recently reached clinical trials. Although the ON-bipolar cells, the first retinal interneurons, make the most attractive targets for optogenetic vision restoration, they have remained inaccessible to human gene therapy due to the lack of a robust cell-specific promoter. We describe the design and functional evaluation of 770En_454P(h
GRM6), a human GRM6 gene-derived, short promoter that drives strong and highly specific expression in both the rod- and cone-type ON-bipolar cells of the human retina. Expression also in cone-type ON-bipolar cells is of importance, since the cone-dominated macula mediates high-acuity vision and is the primary target of gene therapies. 770En_454P(h GRM6)-driven middle-wave opsin expression in ON-bipolar cells achieved lasting restoration of high visual acuity in the rd1 mouse model of late retinal degeneration. The new promoter enables precise manipulation of the inner retinal network and paves the way for clinical application of gene therapies for high-resolution optogenetic vision restoration, raising hopes of significantly improving the life quality of people suffering from blindness.
bipolar cells; cone opsin; gene therapy; human retina; optogenetics; promoter; retinal degeneration; vision restoration.
© 2020 The Author(s).
ON-Bipolar Cells as Therapeutic Targets in Retinal Degeneration (A) Schematic of a healthy retina on the left and a degenerated retina lacking photoreceptors on the right. PR, photoreceptor; HC, horizontal cell; OBC, ON-bipolar cell; OFFBC, OFF-bipolar cell; AC, amacrine cell; GC, ganglion cell. In retinal degeneration, the most distally remaining cells of the visual pathway are the OBCs, which can be engineered into “replacement photoreceptors” by an optogenetic gene therapy. (B) The mGluR6 signalosome in the dendritic tips of OBCs and proteins therein that are affected by loss-of-function mutations—indicated with stars—in congenital stationary night blindness. OBC-targeted gene supplementation therapies can restore function. mGluR6 is a metabotropic glutamate receptor that activates trimeric Gao/Gb3/Gy13 gating the cation channel TRPM1. LRIT3 is required for proper localization of TRPM1, and GPR179 and NYX are scaffolding proteins.
Grm6 Transcription throughout Degeneration in the rd1 Mouse Model Grm6 transcription quantified by quantitative real-time PCR in whole rd1 retinas (n = 6 at each time point) relative to Grm6 transcripts in wild-type C57BL/6 mouse retinas (n = 4). Measured values (●); rod degeneration corrected values (⋄). Grm6 transcription starts decreasing beyond P21, following a power function, R 2 = 0.9866, and stabilizing around 40% of initial Grm6 transcription in late degeneration. ERG, electroretinogram. Data are represented as mean ± SD of biological replicates.
Design of Short Promoters based on Human
GRM6 (A) Genomic region upstream of the translation start site (TLSS) showing the two selected proximal promoter variants, 566P and 454P. (B) Distal genomic region showing the two selected enhancer elements, 407En and 770En. Shaded regions indicate high sequence conservation between the murine and human genomes. Sequence locations are given relative to the TLSS. Markers for regulatory elements: DNase I hypersensitivity clusters, H3K27 acetylation marks, sequence conservation across 100 vertebrates (100 Vert. Cons) and ChIP-Seq peaks. TSS, transcription start site. Images were downloaded from the University of California, Santa Cruz (UCSC), Genome Browser at https://genome.ucsc.edu/ and modified.
Functionality and ON-Bipolar Cell-Type Preference of Novel Synthetic Promoters in Post-mortem HREs (A) Vertical cryosections through the mid-periphery of the human retina transduced with 770En_454P(h
GRM6)-mCitrine. The nuclear stain DAPI differentiates the cell layers, the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL). In this example, 83% of ON-bipolar cells (OBCs) express the reporter mCitrine. Scale bar, 80 μm. (B) Quantification of the promoter strengths by measuring the fluorescence of immunolabeled mCitrine in expressing OBCs. Statistical analysis by one-way ANOVA with post hoc Tukey HSD test; ∗∗p < 0.01; n.s., p > 0.05, not significant. (C) As in (A), but triple staining allowing distinction between rOBCs and cOBCs. In this example, 66% of OBCs express mCitrine. The magnified inserts show the differential labeling of rOBCs (Gao+, PKCa+) and cOBCs (Gao+, PKCa−). Expression efficacies, after Equation 1, and bipolar cell type preferences, after Equation 2, have been calculated for the given example. Scale bar, 50 μm. (D) Quantification of the overall cOBC preference of promoters 770En_454P(h GRM6) (n = 6) and 407En_454P(h GRM6) (n = 3). Statistical analysis by Welch’s t test; ∗∗p < 0.01. Data are represented as mean ± SD of biological replicates.
GRM6) Effectively Targets cOBCs of the Human Macula (A) Terminology of macular areas with location of image acquisition indicated in (B)–(D). (B and C) Foveal (B) and para-foveal (C) sections. Scale bars, 50 μm. (D) Foveal flat mount. Scale bar, 200 μm. (E) Magnification of area indicated in (D). Scale bar, 50 μm. Local ON-bipolar cell expression efficacies are given in percentages. INL, inner nuclear layer.
Capsid-Independent and Widespread ON-Bipolar Cell-Specific Expression Driven by Promoter 770En_454P(h
GRM6) in HREs (A) Flat mount transduced with scAAV2(7m8)-770En_454P(h GRM6)-mCitrine, with the application site indicated with blue arrowheads pointing toward pipette marks. mCitrine indicates that the drop of viral solution remained locally at the application site and, consequently, accessed the inner nuclear layer (INL) predominantly through the inner limiting membrane. Red arrowhead indicates blood vessel. Scale bar, 500 μm. (B–F) The ON-bipolar cell (OBC) expression efficacy, given in percentages, remained approximately constant throughout the explant (B–E), throughout the inner edge (C), middle (D) and outer edge (E) of the INL, and in explants from different donors (A–E versus F). IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bars: 100 μm in (B) and 50 μm in (C)–(F). Staining in (B)–(F) was done with anti-Gao and anti-mCitrine. (G) Transduction fingerprint shown to be independent of the viral capsid used. AAV2(7m8) (n = 3) and AAV2(Y252,272,444,500,700,730F) (n = 3) produced virtually identical expression profiles with a high OBC specificity of ∼88%. Off-target INL includes OFF-bipolar cells and amacrine cells. GCL, ganglion cell layer; ONL, outer nuclear layer. (H) Somatic fluorescence intensities produced by 770En_454P(h GRM6) quantified in immunolabeled cryosections (n = 3). The fluorescence intensities in rOBCs and cOBCs were significantly higher than the intensities in off-target populations. Statistical analysis by one-way ANOVA with post hoc Tukey HSD test: ∗p < 0.05; ∗∗p < 0.01. Data are represented as mean ± SD of biological replicates.
GRM6) Drives Stable, Functional, and Widespread Opn1mw Expression in Late Degenerated rd1 Mice (A) AAV2(7m8)-770En_454P(h GRM6)- Opn1mw-IRES2-TurboFP635-injected rd1 mice were tested for visual acuity determined by the optomotor reflex between 28 and 38 weeks of age (n = 3) and compared to their untreated littermates (n = 5). Optogenetically treated mice showed significant restoration of visual acuity with a restoration of 65% of the visual acuity of wild-type mice (C57BL/6; n = 10). (B and C) Retinas of the treated animals were immunohistochemically evaluated for their expression profiles, as indicated in the whole mounts of (B) at 41 weeks of age and (C) at 26 weeks of age. Scale bars: 1,000 μm in (B) and 50 μm in (C). (D) 770En_454P(h GRM6) had a significant specificity for ON-bipolar cells (OBCs) also in late degenerated tissue at 26 weeks of age. INL, inner nuclear layer; GCL, ganglion cell layer. Statistical analysis by one-way ANOVA with post hoc Tukey HSD test: ∗p < 0.05; ∗∗∗p < 0.001. Data are represented as mean ± SD of biological replicates.
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