Usher syndrome is the most frequent cause of deafness and blindness in humans. It is classified under three clinical subtypes (USH-1, -2 and -3) according to the severity of the symptoms. Approximately 2/3 of patients with Usher syndrome suffer from USH2, 85% of whom have mutations in the USH2A gene. USH2 is clinically characterized by moderate to severe congenital, progressive hearing impairment and retinitis pigmentosa leading to blindness in early childhood to late adulthood. While there is no cure for Usher syndrome, early gene therapy treatment may be viable option to halt the progression of the disease. We have recently demonstrated successful restoration of auditory and vestibular function in a mouse model of USH1C using adeno-associated viral vectors (AAVs) injected through the round window membrane (RWM) at early postnatal age (Pan et al., 2017). This work offers proof-of-principal that the approach is viable for treatment of inherited hearing loss. In this proposal, we take a multi-angle, team lead approach to use stat-of-the-art technologies to develop therapeutic strategies and test their efficacy in halting the progression of hearing loss in several animal models of the disease associated with the most common mutation in the USH2A gene. This mutation, a single base-pair deletion in exon 13 (c.2299delG: p.Glu767Serfs*21) leads to a premature termination codon. Because the USH2A coding sequence extends beyond the capacity for AAV vectors, we propose alternative approaches to develop gene therapy treatments for USH2A. We will assess these therapies in mouse models for this mutation including the c.2299delG (Ush2adelG/delG) which we have partially characterized to show progressive deafness and late-onset visual loss associated with structural defects in the cochlea along with retinal degeneration. We are also in the process of generating a delG swine model to perform pre-clinical studies.
In aim1, we will assess gene supplementation strategies using high capacity adenoviral vectors (AdV) or DNA nanoparticles (NP) to mediate full phenotypic rescue when injected into the inner ear of mutant mice. Multiply-deleted (-E1a/b, -E3, -pol, -pTP) AdV are well suited for this approach due to their high capacity (>15kb) and ability to transduce hair cells successfully both in vitro and in vivo. NP are ideal complement to AdV especially for delivery of large gene as they have been demonstrated to successfully transduce photoreceptor cells of the retina. These vectors will be injected into the inner ear of newborn Ush2adelG/delG mice and longevity of the functional/structural rescue will be evaluated at different times post treatment.
Aim2 will assess the efficacy of gene silencing strategies to correct the c.2299delG mutation. These will include exon skipping strategies using CRISPR/Cas9 constructs as well as antisense oligonucleotides (ASO). To assess and validate gene therapy strategies for future treatment of USH2A, Aim 3 will generate and characterize the auditory phenotype in miniature pig model with the c.2299delG mutation and test the efficacy of viral and non-viral vectors to deliver usherin gene to hair cells and the preservation of auditory sensitivity. In summary, this proposal will provide a solid foundation for development of gene therapy to treat genetic deafness in human.
Usher's syndrome is a devastating disease that destroys auditory and visual function, the two most critical senses that allows interactions with the external world, and to date, there is no cure for it. Studies are planned to evaluate several gene therapy strategies that allow the delivery of large gene or the correction of the defect to prevent hearing loss in models of Usher syndrome type 2a. We have generated several mouse models for this study and are generating a pig model that is essential for preclinical assessments of our therapeutic attempts.