Gene therapy for hereditary hearing loss

Abstract

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...

The role of gene GJB2 and connexin 26 in hearing impairment

“…In order to treat this condition, conducting further research on using interference RNA and gene therapy is highly recommended. According to scien tific literature, this was successfully attempted on transgenic mice and it has resulted in promising results [21]. Using small interference RNA mechanism, the target mRNA, Cys169Tyr Gjb2 in this case is degraded in the cytoplasm after transcription and so down regulating the gene expression [22].…”

“…The viral packaging and delivery of miRNA to Gjb2 Cys-169Tyr human embryos would result in long-term silencing of the Cys169Tyr genes. In addition, the best proposed vector is "bovine adeno associated virus'', which is a single stranded DNA virus and has been shown to be very efficient in gene transfer, as well as relatively safe for causing no cytotoxicity according to previous work done by researchers [21]. The BAA viruses could be then injected to the human embryonic cells in the uterus through microinjec tions methods at suitable time (Before the cochlea develops).…”

“…The BAA viruses could be then injected to the human embryonic cells in the uterus through microinjec tions methods at suitable time (Before the cochlea develops). Finally, the expression of the genes can be assessed using western blots and the newborn hearing processes using 'Auditory brainstem responses' [21,22]. For this reason, advanced research on orphan diseases such as inherited deafness due to GJB2 mutations using gene therapy and other molecular techniques is highly encouraged.…”