Early Disruption of Photoreceptor Cell Architecture and Loss of Vision in a Humanized Pig Model of Usher Syndrome (2022)
Figure 7

Figure 7. Therapeutic approaches.

(A) AAV transduction of retinal layers. AAV8, AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo. Upper Panel: Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples. Dashed circles indicate bleb size. Lower panel: Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer (OS, IS and ONL) for all AAV. Increased exposure (4x) documented transduction of MGC (arrows) by AAV9 and Anc80 but not by AAV8 (representative images from 1 pig, 1 retina each, 2 TR, scale bars 25 µm).

(B) CRISPR/Cas-mediated gene repair was tested in in primary cells of USH1C pigs (see also Figure EV4). Upper left: The cutting site (red arrow) of the most favorable gRNA was directly at the transition from exon 2 (pink) to the downstream intron. Positions of the correcting mutation (blue) and blocking mutation (magenta) are indicated. Lower left: Distinct ssODN binding to the sense (green) or anti-sense strand (orange) were compared. Upper right: Sanger electropherograms confirmed the correct introduction of the correcting (blue) and blocking (magenta) mutations and indicated bystanding NHEJ mutations (grey, overlapping nucleotide peaks) at the gRNA cutting site. Lower right: Frequencies of HDR (blue) and NHEJ (grey) in mixed cell populations were similar for the tested ssODN templates (left). 68 single cell clones treated with urt1.3, indicated that HDR appeared either exclusively (dark blue) or was accompanied by intronic (blue/white shaded) or exonic (blue/gold) mutations (right). In some clones, HDR transformed only the blocking mutation site which is more closely located to the cutting site (white/gold). NHEJ events appeared as insertions (dark grey) or deletions (light grey). The frequency of unmodified clones is depicted in brown.

(C) Effects of harmonin_a1 expression were investigated in dermal fibroblasts. Left panel: Transfection of USH1C primary cells with an expression plasmid (USH1C+) increased the number of ciliated cells towards WT level (3 TR, number of examined cells is indicated, error bars indicate SD, two-tailed Student's t test, *p<0.05). Middle and right panel: Length of cilia in USH1C primary cells was shifted towards WT level (3 TR, number of examined cells is indicated, error bars indicate SD, Kolmogorov-Smirnov-test, *p<0.05, ****p<0.0001).

(D) Efficacy of AAV gene therapy was examined in vivo by sub-retinal injection of Anc80 virus capsid expressing harmonin_a1 under a ubiquitous promoter into a USH1C pig (see also Figure EV5). Left panel: Sub-retinal injection reconstituted harmonin expression in the AAV-treated eye but not in the sham-treated eye (3 TR). Protein extract from a WT retina served as positive control, actin staining as loading control. The 72kD band correlates with harmonin_a isoforms while higher MW indicates putative dimer formation. Middle panel: Immunofluorescence reveals absence of harmonin in the retina of the PBS-injected control eye and harmonin abundance (green) in the retina of the AAV-treated eye. (Representative image from a single animal. Blue: DAPI, scale bar 25 µm). Right panel: Photopic gfERG reveal increased a- and b-wave response to single flash and flicker stimulation in the AAV-treated (red), compared to the sham-treated (black) eyes of two USH1C pigs.

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