Present Molecular Limitations of ON-Bipolar Cell Targeted Gene Therapy

Abstract

Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.

Keywords: AAV vectors; bipolar cells; expression pattern analysis; gene therapy; human retina; optogenetic vision recovery; rd1 mouse model; rd10 mouse model.

Figure 1

Intravitreal injections of AAV7m8 and AAVBP2 into wild-type mice mediate panretinal expression of Opto-mGluR6_IRES_mCitrine under the 4xGRM6-SV40 promoter. (A–C) Retinal in-vivo fundus fluorescence imaging of a control eye (A), and eyes injected with AAV7m8 (B), and AAVBP2 (C), respectively, 3 weeks post injection. The retinas of the treated eyes show a large overall increase in fluorescence, with individual fluorescent cells often visible as bright specks. (D,E) Laser scanning micrographs of AAV7m8 (D) and AAVBP2 (E) treated retinal whole-mounts where mCitrine was labeled immunocytochemically. Despite more intense staining in some regions, mCitrine labeled cells are seen in all areas. (F) A higher magnification of the region indicated by the broken square in (D).

Figure 2

Intravitreal injection of AAV7m8 in wild-type mice primarily expresses Opto-mGluR6_IRES_mCitrine in RBCs and AII amacrine cells when using the 4xGRM6-SV40 promoter. (A) Transverse cryosection of an AAV7m8-treated retina with staining against PKCα (red, marker for RBCs) and mCitrine (green). Primarily two cell populations are labeled including the RBCs and a second layer of cell bodies in the amacrine cell (AC) layer. (B–E) Optical en-face sections of retinal whole-mounts labeled against PKCα and mCitrine taken at different depths from the same area using the same laser microscope settings. (B) Bipolar cell layer: most rod RBCs express mCitrine, with virtually no labeling in cells negative for PKCα. (C) Amacrine cell (AC) layer, a mosaic of brightly transduced cell bodies (green) and the axons of the RBCs (red) are seen. (D) Inner border of the inner plexiform layer (IPL), the fuzzy green background indicates the dendrites of the labeled ACs between the RBC terminals in red. (E) Weak mCitrine staining can be seen in some GCs. The secondary anti-mouse Cy3 antibody also labels blood vessels (red). (F) Triple labeling against mCitrine (blue), PKCα (green), and Gγ13 (red) confirms that transduced BPCs are largely RBCs (yellow) with nearly no labeling in cone OBCs that express Gγ13 but no PKCα (red; marked by arrowheads). (G) The labeled ACs (green) have the AII AC morphology with lobular processes in the OFF-sublamina of the IPL and more extensive dendritic arbors in the ON-sublamina including multiple close contacts with the axon terminals of PKCα-positive RBCs (red). (H) The amacrine cells expressing mCitrine (green) show GLYT1 reactivity (red) in the membranes of their cell bodies and their lobular processes in the OFF-sublamina. The insert shows a magnification of the broken square. (I) A micrograph with the same labeling as in (F) but treated with AAV7m8 (Y444F) instead of AAV7m8. The staining patter is similar, with mCitrine staining primarily restricted to RBCs and a single layer of ACs.

Figure 3

Intravitreal injection of AAVBP2 in wild-type mice primarily expresses Opto-mGluR6_IRES_mCitrine in RBCs and several types of amacrine cells under the 4xGRM6-SV40 promoter. (A) A cryosection labeled against PKCα (red) and mCitrine (green). Primarily two layers of cell bodies are labeled, RBCs and ACs with sparser labeling of GCs. The arrow shows a bright layer of dendrites in the middle of the IPL. (B–E) Optical en-face sections taken at different depths using the same laser microscope settings from the same area stained for PKCα (red) and mCitrine (green). (B) Bipolar cell layer: most RBCs express mCitrine. mCitrine labeling is, however, also seen in cell bodies not positive for PKCα. Horizontal cell processes can also be seen on this micrograph (arrows). (C) Amacrine cell layer: many cell bodies are labeled and the axons of RBCs are seen in red. (D) Middle of the IPL: a network of long beaded dendrites from wide-field amacrine cells is visible. (E) Weak mCitrine staining can be seen in some cell bodies in the GC layer. The secondary anti-mouse Cy3 antibody also labels the blood vessels (red). (F) Triple labeling against mCitrine (blue), PKCα (green), and Gγ13 (red) shows that transfected BPCs are largely RBCs (yellow) with nearly no labeling in cone OBCs that express Gγ13 but no PKCα (marked by arrow heads). The only weakly labeled cone OBC is indicated by a red arrow. (G) Most of the amacrine cells labeled in AAVBP2 retinas (green) express GLYT1 (red) in their cell membranes. (H) Example of a brightly labeled wide-field amacrine cell that projects long beaded dendrites to the center of the IPL (green).

Figure 4

AAV7m8 and AAVBP2 expression patterns in degenerating mouse lines. (A,B) rd1 mice injected with AAV7m8 at the age of 3.5 weeks show in hardly any mCitrine signal (green) in OBCs labeled with Gγ13 (A; red), while most mCitrine positive cells express GLYT1 (B; red). (C,D) As in (A,B), rd1 mice injected with AAVBP2 at the age of 3.5 weeks show little mCitrine expression in OBCs (C) compared to glycinergic amacrine cells (D). (E–H) Intensity profiles of mCitrine expression in AAV7m8 (E) and AAVBP2 (F) treated wild-type retinas and in rd1 AAV7m8 (G) and AAVBP2 (H) treated retinas. In (E,F) the mCitrine profiles (green) show three clear peaks in the GC/IPL, AC and BC layers. For reference, the PKCα signal is indicated in red, with two peaks indicating the cell bodies and axon terminals of the RBCs. Each figure shows the intensity profiles of six micrographs taken from three retinas (broken lines). The averaged signals are indicated in bold. (G,H) Analog intensity profiles of mCitrine expression in rd1 retinas treated with AAV7m8 (G) and AAVBP2 (H). The mCitrine signals in BCs are markedly reduced in the rd1 retinas. (I,J) In the rd10 retina AAVBP2 labels both, BPCs, and ACs similar to the wild-type retina. A large fraction of mCitrine-labeled cells (green) in the rd10 retina express PKCα (I; red) and Goα (J; red).

Figure 5

Transduction patterns of AAV7m8, AAVBP2, and AAV7m8 (Y444F) in human retinal explants. (A–F) Vertical cryosections of human retinal explants transduced with AAV7m8 (A,B), AAVBP2 (C,D), and AAV7m8 (Y444F) (E,F) were labeled with antibodies against mCitrine (green) and Goα (A,C,E, red) or PKCα (B,D,F, red). A similar expression pattern is observed in retinas treated with all three capsids: Opto-mGluR6_IRES_mCitrine is preferentially expressed in the INL, with high expression in OBCs (Goα-positive cells) but also in Goα-negative cells. (G,H) Whole-mount images at the level of the INL of explants treated with AAV7m8 (G) and AAVBP2 (H) stained for mCitrine (green) and PKCα (red). (I,J) Intensity profiles of mCitrine expression in AAV7m8 (I) and AAVBP2 (J) treated explants. For reference, the PKCα signal is indicated in red. mCitrine profiles (green) show a similar distribution, with a large peak in the INL and a second large peak in the ONL which gradually increases toward the outer ONL. A double peak in the INL shows labeling both in the AC layer (first peak) and in the BC layer (second peak). Each figure shows the intensity profiles of 4 images taken from immune-labeled transverse cryosections of two explants (broken lines). The averaged signals are indicated in bold.

Figure 6

Cone OBCs near the fovea centralis express the transgene in human retinal explants transduced with AAV7m8 and AAVBP2. (A–F) Vertical cryosections of human retinal explants transduced with AAV7m8 (A–C) or AAVBP2 (D–F). (A,D) Labeling against Goα (red) and mCitrine (green) near the fovea centralis shows transgene expression in OBCs. (B,E) Labeling against PKCα (red) and Gγ13 (green) of the same regions of neighboring sections to (A) and (D) show that no cells in this region were labeled by PKCα, confirming their cone OBC identity. (C,F) In comparison to (B,E), sections from the mid-periphery show a high fraction of Gγ13-positive cells (green), which also express PKCα (red). OBCs not positive for PKCα are indicated by arrows.