Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter

Abstract

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (~100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.

Keywords: adeno‐associated virus; capsid library; multi‐electrode array; optogenetics; promoter optimization.

Figure 1. AAV8 viral library preparation

1. A The secondary structure of the AAV capsid 8 (PDB:2QA0) with the region targeted for mutation highlighted in red.

2. B Electropherogram of AAV8 capsid sequence from nucleotide 1731 to 1800 containing the mutated region between amino acids 585 and 593.

3. C The schema for AAV capsid variant isolation: In round 1, the AAV library, “AAV2/8lib-dsRed” was subretinally injected into GRM6-GFP mice. After 4 weeks, the retinas were dissociated and green cells isolated by FACS. In round 2, the viral library filtered through round 1 was used to create a titrated AAV2/8lib-dsRed library R1. This was mixed with a non-mutated AAV2/8-dsRed, serving as competing virus, and the viral mix cosubretinally injected into GRM6-GFP mice. From round 2, green/red and red-only cells were isolated by FACS for further analysis of sequence variants.

4. D The electropherograms of AAV capsid sequences amplified by PCR from the cells with only non-mutated sequences isolated from red-only cells (a) and non-mutated as well as variant sequences present in the red/green population (b).

5. E, F The titers of the novel viruses were determined by luciferase assay for small-scale preparations (E) and RT-qPCR for large-scale preparations (F).

Figure 2. Analysis of transduction properties of AAV2/8BP2 virus

1. A, B Representative 20× confocal images of immunostained vibratome sections from retinas of mice subretinally injected (A) or intravitreally injected (B) with viruses. The upper panels show AAV2/8(EF1α-EGFP) injected retinas, and the lower panels show AAV2/8BP2(EF1α-EGFP) injected retinas. The retinas are stained for EGFP (green), cell nuclei (gray), and for the inner plexiform layer strata using choline acetyltransferase (ChAT) (magenta). POS, photoreceptor outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

2. C The number of genome copies per cell was estimated for purified AAV2/8BP2 compared to AAV2/8 following transduction of HEK293 cells.

3. D Cell counts from FACS analysis of retinas from mice (n = 4) subretinally injected with AAV2/8(EF1α-EGFP) or AAV2/8BP2(EF1α-EGFP).

4. E RT-qPCR on RNA from the sorted cells used to determine bipolar cell gene expression levels with 120% increase in Grm6 expression (P = 0.05) and 67% increase in TrpM1L expression (P = 0.04) in the AAV2/8BP2(EF1α-EGFP) cell pool.

5. F Equivalent expression levels measured between cell pools for the cone photoreceptor genes Opnmwl and Opnswl.

6. G Representative 40× confocal images of sections from the retinas of mice subretinally injected with AAV2/8(4 × GRM6-EGFP) (upper panel) and AAV2/8BP2(4 × GRM6-EGFP) (lower panel). The panel on the left shows sections stained for EGFP (green) and cell nuclei (blue), while the panel on the right shows live fluorescence images.

7. H Retinas from mice that were intravitreally injected were similarly analyzed.

Figure 3. Quantitative analysis of transduction properties of AAV8BP2 with a bipolar-cell-specific promoter construct

1. A 10× images of sections from WT mice subretinally injected with AAV2/8(4 × GRM6-EGFP) and AAV2/8BP2(4 × GRM6-EGFP). The sections were stained for EGFP protein expression (green) and for the cell nuclei (blue).

2. B Unfixed and unstained whole-mount images of the cell body, axonal and dendritic regions of the bipolar cells from the injected retinas taken at 40× magnification.

3. C The fluorescent spot counts on local z-stack projections spanning the cell body of these retinas (n = 6, P = 0.01).

4. D–F Cell counts of colocalization of the red (TrpM1L) and green (EGFP) channels (n = 4), with the counts normalized to total red cells (D, P = 0.04) or to total green cells (E). A representative image of the labeled retinal sections used for cell counting is shown (F).

5. G FACS analysis of subretinally and intravitreally injected retinas carried out at 3 weeks post-injection. Representative dot-plot of the fluorescence intensity range for the subretinally injected retinas with GFP versus APC-a (n = 6). AAV2/8 injected retina is shown in the top panel while AAV2/8BP2-injected retina is shown in the bottom panel.

6. H The FACS intensity range divided across seven regions, Fr8 to Fr14, with the percentage of fluorescent cells shown on the y-axis.

7. I A representative 40× image of a section from subretinally injected retinas stained for EGFP (green), cell nuclei (blue), and the rod-bipolar cell marker PKCα (red).

8. J Expression of kcng4, lhx4, prkcα, and grm6 in cells of sorted fractions Fr12 to Fr14 (highlighted by a red box in the upper panel of Fig 3H) relative to expression in unsorted cells from total retina.

Figure 4. Light responses in rd1 mice after transducing with AAV2/8BP2 expressing CatCh

1. Light responses to full field stimulation from six example cells. Both ON, OFF and ON-OFF cells are recorded.

2. Histogram of peak firing frequencies.

3. Response to increasing spots.

4. Firing rate as a function of light intensity.