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Dissertation
Treating the cell: advancing gene therapy for hearing loss
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2017
DOI: 10.17077/etd.005242
Abstract
Hearing impairment is the most common sensory deficit, estimated to impact 23% of Americans over age 12 and 46% of Americans over age 48. Despite its high prevalence, there are no biological treatments for sensorineural hearing loss. This is due, in part to our limited understanding of the complex microarchitecture of the inner ear, which contains highly specialized sensory and supporting cells that differ widely in morphology and function. In a striking example of cellular cooperation, these cells function in concert to facilitate the perception of sound. Due to the complex nature of this system -- with many critical anatomical structures, distinct cell types, and molecular level components -- a wide variety of disruptions can cause the system to fail resulting in a hearing loss phenotype. The identification of deafness genes and individual causative variants has enabled the design of gene therapy strategies, capable of repairing these genetic defects. Similarly, studies aimed at dissecting the molecular pathways critical to hearing function and hair cell survival at the single cell level have the potential to inform the design of novel therapeutic strategies.
Herein, we describe our efforts to design a broadly applicable gene therapy strategy and to better characterize the critical cell types of the inner ear at the single cell level. The goal of these studies is to further our understanding of the critical cells in the auditory system and leverage that information to develop gene therapy strategies for hearing loss and deafness.
Toward these goals, we have designed and tested a miRNA construct packaged inside an AAV2/9 viral vector that specifically targets and knocks down the deafness-causing Beethoven mutation in the heterozygous (Bth/+) Beethoven mouse model of dominant progressive hearing loss. Delivery of this construct at postnatal day 2 (p2) by intracochlear round window membrane (RWM) injection slowed progression of deafness up to 35 weeks. We have built on this initial success by redesigning the therapeutic construct, enabling the application of a single construct to any variant in the Tmc1 gene. Importantly, this approach can be adapted to other deafness genes and utilized for both dominant and recessive forms of genetic deafness.
To better characterize mature auditory hair cells, we have developed and implemented a strategy for single cell gene expression profiling of mature cochlear cell types. We have utilized this approach to profile gene expression from individual outer hair cells (OHCs), inner hair cells (IHCs), and Deiters’ cells (DCs). This work has identified novel marker genes of auditory hair cells, as well as temporal- and position-based differential expression patterns in the murine cochlea. Furthermore, this single cell RNA- Seq dataset has revealed 17 novel exonic regions in 12 deafness causing genes, and suggests the expression of hundreds of novel isoforms resulting from previously unappreciated splicing diversity.
Details
- Title: Subtitle
- Treating the cell: advancing gene therapy for hearing loss
- Creators
- Paul Thomas Ranum
- Contributors
- Richard J.H. Smith (Advisor)Botond Banfi (Committee Member)Robert A Cornell (Committee Member)Beverly L Davidson (Committee Member)Val C Sheffield (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular and Cellular Biology
- Date degree season
- Autumn 2017
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.005242
- Number of pages
- xvi, 146 pages
- Copyright
- Copyright 2017 Paul Thomas Ranum
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 140-146).
- Academic Unit
- Interdisciplinary Graduate Program in Molecular Medicine
- Record Identifier
- 9983779600002771
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