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Maryna Ivanchenko, Daniel Hathaway, Eric Mulhall, Mantian Wang, Alex Klein, Yaqiao Li, Bence György, David Corey; Development of Dual-PCDH15 AAV Gene Therapy for Usher Syndrome Type 1F Blindness and Deafness. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1110.
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© ARVO (1962-2015); The Authors (2016-present)
Usher 1F is caused by mutations in the PCDH15 gene, which encodes the tip-link protein PCDH15 in the inner ear. In the eye it has been shown to localize to the calyceal processes of the photoreceptor. Usher 1F is characterized by profound congenital deafness, and by progressive blindness beginning in the second decade. Gene addition therapy could be an attractive treatment, however, the PCDH15 coding sequence of ~5.8 kb is too large to fit into a single AAV capsid. We used a dual-AAV strategy to circumvent the size limitation.
We engineered two vectors that each encode part of the full-length protein. In cells, AAV genomes can recombine to creat a full-length coding sequence. To evaluate function in cochlea in vivo, Pcdh15 cKO mice were treated with dual AAVs. Hearing tests and histological analyses were performed at P30. To assess the potential of dual-AAV gene expression in human retina in vitro, we transduced retinal organoids from human iPSCs with dual AAVs and five weeks later evaluated HA-tagged PCDH15 expression and localization with immunofluorescence and immunogold SEM imaging.
In HEK cells, full length PCDH15 was successfully produced using dual-AAV delivery. Proper recombination and splicing were confirmed with RT-PCR and Sanger sequencing. We found that Pcdh15 cKO mice, injected into inner ear with dual AAVs, displayed tip links four weeks after injection. They also showed robust rescue of hair bundle morphology, and rescue of mechanotransduction. While uninjected Pcdh15 cKO mice were deaf and had degenerated hair bundles, mice treated with dual AAVs encoding PCDH15 demonstrated good hearing rescue.We also characterized the ultrastructure of photoreceptors in retinal organoids. SEM showed that a majority of photoreceptors developed inner segments, while some formed outer segments and connecting cilia. We observed nascent calycal processes at the apical ends of inner segments. Retinal organoids from human iPSCs transduced with dual AAVs encoding HA-tagged PCDH15 showed antibody labeling of HA-tag in photoreceptors, which was localized on the surface of the inner segments and at the inner/outer-segment junction where calyceal processes develop.
Dual-AAV delivery of PCDH15 mostly restores hearing in a mouse model of Usher 1F, and mediates expression and normal localization of PCDH15 in human photoreceptors in vitro. It holds promise for treatment of Usher 1F.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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