Usher syndrome is a devastating autosomal recessive disorder that causes hearing loss and blindness. While cochlear implants have been able to offer prognosis for the hearing loss, there is currently no available treatment that can alleviate or cure the visual symptoms. Gene replacement therapy using recombinant adeno-associated viruses (AAVs) is a promising approach to restore gene expression in the retina; however, the relatively small capacity of AAVs prevents the delivery of large genes, such as those associated with Usher syndrome. To overcome this, genes can be split between two separate vectors, which can then reconstitute post-transduction. In this study, we investigated gene replacement therapy via dual AAV transduction for the prevention of retinal degeneration in Usher type 1F, caused by a mutation in the PCDH15 gene. PCDH15 was split into left and right halves and packaged into AAV2/9 and AAV2/Anc80L65 serotypes. Dual AAV vectors were tested in vitro in i) patient fibroblasts and ii) patient and control line retinal organoids (ROs) derived from induced pluripotent stem cells (iPSCs). Transduction with dual AAVs containing the separate halves of PCDH15 successfully induced PCDH15 transcript expression in patient fibroblasts, but RO transductions yielded variable results. Of the vector strategies tested, hybrid Anc80 vectors showed higher expression in fibroblasts while AAV2/9 vectors resulted in the highest fold increase in PCDH15 transcript expression in patient ROs. Our results demonstrate proof-of-principle expression restoration of gene expression by dual AAV transduction. However, further work is required to determine the viability of this approach for Usher 1F patients.