Mouse Model Systems to Interrogate Candidate Genes for Sensory Hair Cell Regeneration
John Brigande, Ph.D., Oregon Health & Science University

As HRP scientists detect and characterize genes that are hypothesized to participate in the activation or inhibition of hair cell regeneration, methods for altering or disrupting those genes are critical to the demonstration of their importance. This project aims to couple together two sophisticated methods for manipulating genes in mice, the so-called CRISPR-READI and i-GONAD methods. CRISPR-READI enables efficient, large gene edits, and i-GONAD simplifies the delivery of reagents for gene editing. Together, the proposed CRISPR-READI-GO method should allow for rapid and efficient gene editing to be put to use by HRP investigators.

Mouse Model Systems to Interrogate Candidate Genes for Sensory Hair Cell Regeneration
John Brigande, Ph.D., Oregon Health & Science University

Given what the HRP has discovered thus far, this project proceeds from the assumption that manipulation of multiple genes that regulate diverse signaling pathways may be required to reprogram supporting cells to become functional hair cells. We must first verify the expression pattern of candidate genes in the inner ear, then develop methods that allow us to modulate multiple candidate genes in each supporting cell in order to transmit the genetic instructions that trigger regeneration. This proposal’s first goal is to establish improved genome editing via oviductal nucleic acids delivery (iGONAD) technology, allowing us to identify as well as eliminate candidate genes. Our second goal is to turn on multiple genes simultaneously in the same cell using a mouse model. The overall objective is to establish a rapid method to describe candidate gene expression and function in the inner ear at any stage, and to define an approach to determine which genes are critical to modulate signaling pathways for hair cell regeneration.

Mouse functional testing
John Brigande, Ph.D. Oregon Health & Science University

The conceptual framework of this project wrestles with a persistent challenge facing the HRP consortium: We must verify that the candidate genes we advance as regenerative genes actually perform as advertised. Is our altering of the gene expression of a candidate gene truly the trigger that turns supporting cells into hair cells? Our solution is to devise a mammalian model system that meets several definitive criteria. First, we need deafened adult mammalian inner ears to detect the production of new hair cells; we achieve this genetically by specifically killing hair cells that are uniquely sensitive to a bacterial toxin. Second, we need a way to turn on or turn off the candidate gene after the hair cells are dead; we achieve this by chemically activating a gene that in turn unmasks the expression of the proposed candidate. Third, we need a way to detect newly produced hair cells in the cochlea; we achieve this by using a tissue clearing and staining procedure developed with HRP funding that allows us to detect hair cells produced from supporting cells.

This entire approach is called a model system for validating candidate genes for hair cell regeneration. But one size does not fit all, and we need to continually adapt the core model system to achieve full functionality. In this proposal, we aim to test our model system in healthy ears to see if tweaking our candidate genes can produce hair cells from supporting cells in the absence of widespread hair cell death. The idea here is to make sure that the bacterial toxin–mediated destruction of hair cells is not interfering with our candidate gene activity and new hair cell production. Our second goal is to test a new virus delivery system that will allow us to evaluate larger candidate genes. Presently, we can only express very small genes with the virus we are using, restricting candidate gene verification. Our final goal is to evaluate a modified viral vector that is encased in lipid membranes to learn if it can express candidate genes more efficiently in supporting cells. The benefit of this approach is that viral production is quick, inexpensive, and requires no special training or expertise. Successful completion of this proposal will establish a comprehensive, cost-effective approach to aggressively validate candidate genes for hair cell regeneration.

A mouse model system to interrogate candidate genes for sensory hair cell regeneration
John Brigande, Ph.D. Oregon Health & Science University

This Phase II project involves the delivery of genes and reagents that could regulate hair cell regeneration into the embryonic inner ear of mice. Two approaches will be taken. In the first, transcription-factor genes that activate pathways that may be involved in regeneration will be delivered, and their ability to trigger hair cell regeneration after those cells are killed will be assessed. In the second, CRISPR/Cas9 reagents will be used to turn off genes that might be preventing hair cell regeneration. While these are experimental and not therapeutic approaches, the project will allow us to determine whether any specific molecules are capable of triggering hair cell regeneration in the mouse.

An in vivo model system to interrogate gene and pathway candidates involved in sensory epithelial regeneration of the mammalian inner ear
John Brigande, Ph.D. Oregon Health & Science University

An example of the consortium moving into phase II, this project will deliver into the embryonic inner ear of mice genes that could regulate hair cell regeneration; these genes will be in an inactive state. Delivery of an activating molecule in the mature inner ear will then allow that gene to be expressed an appropriate time relative to hair cell damage. While this delivery method is not appropriate for any eventual therapy, it will allow us to examine to determine whether any specific molecules are capable of triggering hair cell regeneration in the mouse.