Gene Therapy in Mouse Models of Deafness and Balance Dysfunction

Therapeutic strategies to restore hearing and balance in mouse models of inner ear disease aim to rescue sensory function by gene replacement, augmentation, knock down or knock out. Modalities to achieve therapeutic effects have utilized virus-mediated transfer of wild type genes and small interfering ribonucleic acids; systemic and focal administration of antisense oligonucleotides (ASO) and designer small molecules; and lipid-mediated transfer of Cas 9 ribonucleoprotein (RNP) complexes. This work has established that gene or drug administration to the structurally and functionally immature, early neonatal mouse inner ear prior to hearing onset is a prerequisite for the most robust therapeutic responses. These observations may have significant implications for translating mouse inner ear gene therapies to patients. The human fetus hears by gestational week 19, suggesting that a corollary window of therapeutic efficacy closes early in the second trimester of pregnancy. We hypothesize that fetal therapeutics deployed prior to hearing onset may be the most effective approach to preemptively manage genetic mutations that cause deafness and vestibular dysfunction. We assert that gene therapy studies in higher vertebrate model systems with fetal hearing onset and a comparable acoustic range and sensitivity to that of humans are an essential step to safely and effectively translate murine gene therapies to the clinic

Mouse Middle Ear Ion Homeostasis Channels and Intercellular Junctions

The middle ear contains homeostatic mechanisms that control the movement of ions and fluids similar to those present in the inner ear, and are altered during inflammation.The normal middle ear cavity is fluid-free and air-filled to allow for effective sound transmission. Within the inner ear, the regulation of fluid and ion movement is essential for normal auditory and vestibular function. The same ion and fluid channels active in the inner ear may have similar roles with fluid regulation in the middle ear.Middle and inner ears from BALB/c mice were processed for immunohistochemistry of 10 specific ion homeostasis factors to determine if similar transport and barrier mechanisms are present in the tympanic cavity. Examination also was made of BALB/c mice middle ears after transtympanic injection with heat-killed Haemophilus influenza to determine if these channels are impacted by inflammation.The most prominent ion channels in the middle ear included aquaporins 1, 4 and 5, claudin 3, ENaC and Na(+),K(+)-ATPase. Moderate staining was found for GJB2, KCNJ10 and KCNQ1. The inflamed middle ear epithelium showed increased staining due to expected cellular hypertrophy. Localization of ion channels was preserved within the inflamed middle ear epithelium.The middle ear epithelium is a dynamic environment with intrinsic mechanisms for the control of ion and water transport to keep the middle ear clear of fluids. Compromise of these processes during middle ear disease may underlie the accumulation of effusions and suggests they may be a therapeutic target for effusion control

Otitis Media Impacts Hundreds of Mouse Middle and Inner Ear Genes

<div><p>Objective</p><p>Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition.</p><p>Methods</p><p>To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed <i>Hemophilus influenza</i> using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes.</p><p>Results</p><p>Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear <i>up</i>regulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear <i>down</i>regulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear.</p><p>Conclusion</p><p>Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media.</p></div

Middle and inner ear genes affected by acute inner ear inflammation.

<p>Middle and inner ear genes affected by acute inner ear inflammation, >1.5× upregulation or <1.5× downregulation. Note that while there is overlap between the shared inner and middle ear genes up- or downregulated, there is a high percentage of unique genes in both categories.</p

Interactome Analysis of ME and IE Genes.

<p>Interactome analysis: performed with limiting the dataset to named genes and protein/gene interactions <i>in our data set</i>, fold change >1.5× upregulation and <1.5× downregulation and p<0.05.</p

IL-17 pathway map.

<p>IL-17 pathway map, showing all genes in the IL-1 gene pathway and the amount of upregulation seen in our dataset by the histogram bars (G-CSF, GM, CSF, c-FOS, CCL2 and 7, iNOS, GRO-1, IL-2, IL-1, IL-8, RANKL, Stromelysin, JAK2, C/EPbeta). Highest fold change activity seen in IL-6 for both ME and IE. (Data summary from Genego.)</p

Cross comparison of cytokine gene expression measured by Affymetrix Gene Chip and qRT-PCR.

<p>Middle ear fold change analyses (top) showed a significant correlation between the two methods (p = 0.0098). Inner ear analyses (bottom) showed a similar pattern of cytokine expression that also was significantly correlated between the two methods (p = 0.0241).</p

Impact of Middle Ear Inflammation on Ion Homeostasis Genes in ME and IE.

<p>Impact of Middle Ear Inflammation on Ion Homeostasis Genes in ME and IE.</p