Development of gene transfer tools for delivery of MiRNAs to the inner ear
Abstract
Hearing loss affects communication so profoundly that patients often must contend with feelings of loneliness and depression. Deafness can be treated with cochlear implants in many but not all patients, although this technology cannot restore hearing acuity to normal. My research focused on creating and investigating potential therapeutic tools that could restore acuity and detection of sound to people suffering from hearing loss caused by a lack or loss of hair cells. The supporting cells located beneath the hair cells in sensory organs of the inner ear offer an excellent cellular source to target for hair cell regeneration. Hair cells and supporting cells are created from the same progenitor pool during development, and in birds, hair cell loss stimulates supporting cells to divide and regenerate additional hair cells. The factors underlying the fate choice of supporting cell vs. hair cell may provide potential reagents for treating deafness and balance disorders. Previous research has focused on supplying adult supporting cells with the pro-hair cell transcription factor, Atoh1, to force those cells to switch to a hair cell fate, but an alternative approach is to identify and supply anti-supporting cell factors to assist in converting supporting cells to a new fate. Presently, members of the miRNA-183 family are likely candidates to serve as anti-supporting cell signals. Another miRNA, miR-9, may also prove to be influential in discouraging a supporting cell fate by decreasing the levels of a transcription factor, HES-1, that normally negatively regulates the pro-hair gene, Atoh1. I produced two vectors containing the entire miR-183 family within a single artificial intron located upstream of a protein-encoding exon. I demonstrated that this design facilitates the coordinated expression of all three mature miRNAs and the associated protein. When this cassette is placed within mammalian viruses, the viruses can successfully replicate and produce the miRNA and protein in vitro. However, placement of the cassette within an avian virus, RCAS, impedes viral replication and shows minimal production of the cassette products in vivo. In addition to developing different transfer tools, I also showed for the first time with in situ hybridization the localization of miR-9 within the sensory domains of the avian inner ear. Therefore, the role of miR-9 in inner ear development warrants further investigation.
Degree
Ph.D.
Advisors
Fekete, Purdue University.
Subject Area
Molecular biology|Developmental biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.