Development of Gene Therapy for Hereditary Deafness using Rational Protein Engineering - 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.
