PROJECT SUMMARY Deafness and blindness are two of the most common and most devastating neurological disorders. Seldom fatal, they separate patients from the world they live in and the people they love, for a lifetime. In many cases, combined deafness and blindness result from a single gene defect, usually with a mutant gene copy inherited from both parents. Usher syndrome is a devastating hereditary deafness and blindness caused by mutation of any of nine genes. One gene, PCDH15, when mutated, causes Usher syndrome type 1F, manifesting as profound congenital deafness, balance disorder and progressive blindness. There is no treatment. New methods are rapidly being developed for gene therapy to treat monogenic disorders, methods that change patient lives. Here, we propose to develop a gene therapy strategy for Usher Syndrome Type 1F and to initiate its path to the clinic to treat different pathologies of the disease. Adeno-associated virus (AAV) vectors have been found to be efficient and effective for gene therapy in the inner ear and their application in development of gene addition therapies to treat Usher syndrome type 1F is an attractive approach. However, the PCDH15 coding sequence of almost 6 kb is too large to fit in AAV, which has a capacity of ~4.7 kb. Most of PCDH15 resembles a “bead chain” structure of 11 beads, with binding domains at N- and C-terminal ends. We have developed novel “mini-PCDH15” constructs that retain the binding domains but lack 4-5 beads, and that consequently fit in AAV vectors. They show normal binding in vitro; at least two rescue hearing in our Pcdh15-knockout mouse model. We will use these and new mini-PCDH15 constructs to rescue hearing and vestibular function in Pcdh15-knockout mice. We will also employ in silico and in vitro structural biology and protein chemistry to carefully characterize each successful mini-PCDH15, and use that for iterative optimization of engineered mini-PCDH15s. Finally, we will test the best performing mini-PCDH15 for localization and toxicity in non-human primate ear as part of their preclinical evaluation. Because mechanical stress on PCDH15 is greater in the inner ear than in the eye, the engineered mini-PCDH15 constructs that successfully rescue hearing or balance will also have the potential to halt or even reverse the progressive vision loss in patients. In addition, methods we develop in this study using a rational, iterative, structure-based mini-gene approach to develop Usher 1F therapy will be applicable to many other deafness genes with coding sequences that do not fit in AAV. 1

NARRATIVE The advent of successful gene therapy for certain retinal disorders and our extensive knowledge of the dozens of genes involved in hereditary hearing loss, together raise hope that gene therapy can be used to treat genetic forms of hearing loss. For instance mutations in the PCDH15 gene cause Usher syndrome type 1F, a devastating deafness, blindness and balance disorder. While we and others have achieved success in mouse models using gene therapy for short genes that will fit in adeno-associated virus (AAV) vectors, PCDH15 is a long gene that does not fit into AAV; thus we propose to engineer short “mini-PCDH15” proteins, to test rescue of hearing and balance in mice, and to test localization and toxicity in nonhuman primates. 1