Development of effective therapeutics for hearing loss has proven to be a slow and difficult process, evidenced by the lack of restorative medicines and technologies currently available to the otolaryngologist. In large part this is attributable to the limited regenerative potential in cochlear cells and the secondary degeneration of the cochlear architecture that commonly follows sensorineural hearing impairment. Therapeutic advances have been made using animal models, particularly in regeneration and remodeling of spiral ganglion neurons, which retract and die following hair cell loss. Natural regeneration in avian and reptilian systems provides hope that replacement of hair cells is achievable in humans. The most exciting recent advancements in this field have been made in the relatively new areas of cellular replacement and gene therapy. In this review we discuss recent developments in gene- and cell-based therapy for hearing loss, including detailed analysis of therapeutic mechanisms such as RNA interference and stem cell transplantation, as well as in utero delivery to the mammalian inner ear. We explore the advantages and limitations associated with the use of these strategies for inner ear restoration.

Hearing loss is the most common sensory deficit worldwide. It affects ∼5% of the world population, impacts people of all ages, and exacts a significant personal and societal cost. This review presents epidemiological data on hearing loss. We discuss hereditary hearing loss, complex hearing loss with genetic and environmental factors, and hearing loss that is more clearly related to environment. We also discuss the disparity in hearing loss across the world, with more economically developed countries having overall lower rates of hearing loss compared with developing countries, and the opportunity to improve diagnosis, prevention, and treatment of this disorder.

Targeting and down-regulating specific genes with antisense and decoy oligonucleotides, ribozymes or RNA interference (RNAi) offers the theoretical potential of altering a disease phenotype. This article reviews the molecular mechanism behind the in-vivo application of RNAi-mediated gene silencing, focusing on its application to the inner ear. RNAi is a physiological phenomenon in which small, double-stranded RNA molecules (small interfering RNA, siRNA) reduce expression of homologous genes. Notable for its exquisite sequence specificity, it is ideally applied to diseases caused by a gain-of-function mechanism of action. Types of deafness in which gain-of-function mutations are observed include DFNA2 (KCNQ4), DFNA3 (GJB2) and DFNA5 (DFNA5). Several strategies can be used to deliver siRNA into the inner ear, including cationic liposomes, adenoassociated and lentiviral vectors, and adenoviral vectors. Transduction efficiency with cationic liposomes is low and the effect is transient; with adeno-associated and lentiviral vectors, long-term transfection is possible using a small hairpin RNA (shRNA) expression cassette.

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Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause autosomal recessive nonsyndromic hearing loss. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.

Hearing impairment is the most common sensory deficit worldwide, affecting at least one child in every one thousand born. Gene therapy targeting the inner ear offers promise for treatment of genetic forms of hearing loss. Many genetic forms of deafness are congenital and gene therapies in these cases would require treatment prior to inner ear maturation. Included in this category is the dominant-negative R75W mutation in GJB2 which encodes connexin 26, a gap junction protein expressed in the supporting cells of the organ of Corti. RNA interference (RNAi)-based therapeutics offer promise for treating dominant-negative diseases. Our goal has been the in vivo application of RNAitherapy to the GJB2-R75W transgenic mouse, a model of severe-to-profound dominantnegative hearing loss. Here we describe our efforts to identify a therapeutic, a suitable delivery route, and an optimal delivery vector. We have designed and optimized siRNA to achieve robust silencing of the mutant transgene in vitro and have prepared artificial miRNA constructs for in vivo application. We have determined to use the embryonic otocyst microinjection technique as the route for therapeutic delivery and have successfully utilized this technique to study the tropism and safety of several viral vector (adeno-associated virus 2/1, early-and late-generation adenoviruses, and bovine adenoassociated virus). For the first time we have characterized viral tropism for cochlear supporting cells following in utero delivery to their progenitor cells in the developing cochlea and identified bovine adeno-associated virus as a safe vector for gene delivery to the supporting cells of the cochlea. We have also described two previously unreported phenotypes in the GJB2-R75W transgenic mouse model: skin disease and cataracts. Both can be caused by dominant connexin mutations in humans. Our work shows that although gene therapy is not simple, powerful tools are in place for treating dominant forms of hereditary hearing loss. ACKNOWLEDGMENTS A special Thank You to Dr. Richard Smith for his mentorship, leadership, and support; to my Thesis Committee (Drs. Michael Anderson, Terry Braun, Michael Henry, and Michael Welsh) for their encouragement and important role in my education; to members of the Molecular Otolaryngology Research Laboratories for being there each day; to Val Sheffield for his counsel and insights; and also to the Animal Caretakers who made my research possible. I would also like to express my appreciation to the many people who have assisted in this research: Dr. Michael Hildebrand provided supervision and assistance throughout my time in the lab; Dr. Sam Gubbels (University of Wisconsin) taught us the embryonic otocyst injection and continued to provide extensive help with injections; Dr. Marlan Hansen and members of his lab for assistance with microscopy and cochlear dissection; Dr. Frederic Venail for his assistance with viral vectors and cochlear explants; Charles Searby and Qihong Zhang of the Val Sheffield lab for generous and ongoing help with met...

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