PCDH15 Dual-AAV Gene Therapy for Deafness and Blindness in Usher Syndrome Type 1F

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 a novel approach for USH1F gene therapy, exceeding the single AAV packaging limit by employing a dual adeno-associated virus (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 in clinically relevant retinal models, including human retinal organoids and non-human 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.

Optimization strategies and advances in the research and development of AAV‐based gene therapy to deliver large transgenes

“…This technology was developed due to the properties of the AAV genome to be concatemerized in the head‐to‐tail direction 86,91 . Dual AAV vectors have been extensively studied for different experimental and disease modalities (DMD, Usher syndrome, hearing loss, Stargardt disease and dysferlinopathy) 92–101 . Several approaches can be successfully used to assemble large transgene in multiple AAV vectors.…”

“…Upon co‐transfection of cells with dual AAV vectors, the ITRs are concatemerization according to the head‐to‐tail orientation. The SD and SA are located at the ends of cDNA in each AAV vector and are trans‐spliced, followed by the production of full‐sized mRNA and protein 73–102 . The SD and SA sequences for trans‐splicing AAV are vital in promoting the correct trans‐splicing process.…”

“…GFP and RFP) are one the most studied objects for CP. The development of a new bright fluorescent permutant has great potential in basic and translational research including biosensors (Ca2+, Zn2+, Cu2+, NH4+ and voltage probes), optogenetics and in vivo imaging 93,95–130 . Examples of circularly permuted proteins are not limited by fluorescent proteins that hold promises in their broad application in protein engineering and biotechnology (phosphoribosyl anthranilate isomerase from yeast, 129 aspartate transcarbamoylase, 131 the SH3 domain of α‐spectrin, 132 chymotrypsin inhibitor, 133 and thiol/disulfide oxidoreductase DsbA from Escherichia coli 133,134 ; and several other proteins).…”

Elasticity and Thermal Stability are Key Determinants of Hearing Rescue by Mini-Protocadherin-15 Proteins

“…Adeno-associated viruses (AAV) have been shown to be efficient and effective for gene therapy [31][32][33][34][35] , but these have an inadequate packaging capacity (< 4.7 kb 36 ) to carry the full length PCDH15 coding sequence in a single AAV. Dual-AAV approaches have been successfully implemented in several gene therapy studies and clinical trials [37][38][39][40][41][42][43] , including two aiming to express full-length PCDH15 44,45 . However, shortening PCDH15 by removing possibly unnecessary domains could enable a more efficient transgene delivery to patients by a single AAV.…”