PCDH15 dual-AAV gene therapy for deafness and blindness in Usher syndrome type 1F models
Maryna V. Ivanchenko, … , Bence György, David P. Corey
Maryna V. Ivanchenko, … , Bence György, David P. Corey
Published October 23, 2024
Citation Information: J Clin Invest. 2024;134(23):e177700. https://doi.org/10.1172/JCI177700.
View: Text | PDF
Research Article Ophthalmology Otology

PCDH15 dual-AAV gene therapy for deafness and blindness in Usher syndrome type 1F models

Abstract

Usher syndrome type 1F (USH1F), resulting from mutations in the protocadherin-15 (PCDH15) gene, is characterized by congenital lack of hearing and balance, and progressive blindness in the form of retinitis pigmentosa. In this study, we explore an approach for USH1F gene therapy, exceeding the single AAV packaging limit by employing a dual–adeno-associated virus (dual-AAV) strategy to deliver the full-length PCDH15 coding sequence. We demonstrate the efficacy of this strategy in mouse USH1F models, effectively restoring hearing and balance in these mice. Importantly, our approach also proves successful in expressing PCDH15 protein in clinically relevant retinal models, including human retinal organoids and nonhuman primate retina, showing efficient targeting of photoreceptors and proper protein expression in the calyceal processes. This research represents a major step toward advancing gene therapy for USH1F and the multiple challenges of hearing, balance, and vision impairment.

Authors

Maryna V. Ivanchenko, Daniel M. Hathaway, Eric M. Mulhall, Kevin T.A. Booth, Mantian Wang, Cole W. Peters, Alex J. Klein, Xinlan Chen, Yaqiao Li, Bence György, David P. Corey

×

Figure 5

Expression of PCDH15 in human retinal organoids by dual AAV vectors.

Options: View larger image (or click on image) Download as PowerPoint
Expression of PCDH15 in human retinal organoids by dual AAV vectors. (A)...
(A) Organoids were transduced with dual AAV vectors, and 5 weeks later expression was analyzed by antibodies against the HA tag. (B) Representative confocal microscopy images captured from cryosections of non-transduced human retina organoids. Anti-PCDH15 labeling (green) was detected in the photoreceptors, especially at the distal ends of outer segments (OSs) (yellow arrows) (n = 3 organoids). Hoechst dye labeled nuclei, anti–cone arrestin (ARR3) labeled cones, and anti-rhodopsin (Rho) labeled rod OSs. (C) Scanning electron microscopy of human retinal organoids. The finger-like calyceal processes (yellow arrows) protrude from the apical region of the inner segments (ISs) of both rod (middle) and cone (right) photoreceptors (n = 3 organoids). (D) Immunogold scanning electron microscopy labeling of non-transduced human retinal organoids immunostained with anti-PCDH15 primary antibody and 12 nm gold-conjugated secondary antibody. Endogenous PCDH15 was located at the surfaces of ISs and of nascent calyceal processes of photoreceptors (n = 3 organoids). (E) Anti-HA fluorescent labeling of vector-delivered HA.PCDH15. Because the CMV promoter is only present on the 5′ vector, HA.PCDH15 and GFP were only expressed when both the 5′ and 3′ vectors were added to the organoids and were internalized within a cell. Anti-HA.PCDH15 is shown as magenta and GFP as green. In these images, GFP diffusely labeled doubly transduced cells. HA.PCDH15 is seen at the junction of ISs and OSs of cone photoreceptors (n = 3 organoids). (F) Immunogold scanning electron microscopy confirmed localization of HA-tagged hsPCDH15 to calyceal processes and ISs of human retinal organoids transduced with dual-AAV (n = 3). Multiple 12 nm gold beads were detected. Scale bars: 5 μm (B and E), 10 μm (C, left), 1 μm (C, right), 0.5 μm (D and F).