Sensorineural hearing loss (SNHL) is a significant healthcare problem, with large social and financial costs. SNHL occurs when the auditory sensory hair cells of the inner ear, or the primary neural pathways connecting these cells to the brain, cease to function appropriately. Such damage occurs consequent to genetic and/or environmental insults. SNHL is permanent because of subsequent sensory and/or neural cell death, and the inability of such cells to regenerate. There are many ideas about how to provoke regeneration of lost auditory hair cells and neurons in mammals. These include stimulating regeneration of hair cells from residual endogenous supporting cells, as occurs normally in fish and birds. Similarly, spiral ganglion neurons might be regenerated from glia. In both cases, manipulating the signals and transcription factors normally involved in the development of these cells is under intensive investigation. Stem cell-based replacement strategies are also being pursued. Regardless of the preferred approach, any proposed therapy for SNHL will require testing in an in vivo mammalian model. However, existing mammalian models of sensory and neural cell loss in the peripheral auditory system are not ideal. For example, both ototoxic drugs and noise exposure can be controlled temporally to cause hearing loss, but these treatments affect the entire inner ear, not only the auditory hair cells and/or spiral ganglion neurons, and this can confound treatment studies. Inducible single recombinase-based methods of ablating inner ear sensory or neural cells are promising, but also frequently impact other cells/tissues that are essential for viability of the animal, thus limiting their utility. In this application, w propose to develop and validate an improved system, called intersectional and inducible cell-specific ablation (IICSA), for inducing precise and reproducible ablation of mouse auditory hair cells or spiral ganglion neurons at any stage of interest. This will enable modeling of inner hair cell, outer hair cell or spiral ganglion neuron loss at different stages, and provide a platform fo testing hearing restoration therapies in a mammal. IICSA is a significant advance over current technology because it will enable doxycycline-inducible cell ablation with the potential for reduced or absent off-target effects. We will use state- of-the-art targeted transgenesis and genome editing techniques to generate the necessary IICSA driver and effector mouse strains. This technology will be developed and tested for inducible ablation of specific cochlear cell types, but is adaptable to any target cells of interest, enabling modeling of any number of degenerative conditions for both basic and translational studies.
Permanent hearing loss caused by degeneration of inner ear sensory or neural cells affects up to one third of individuals by the age of 80 and generates significant social and healthcare costs. Testing potential drug or cell- based therapies requires mouse models in which relevant inner ear cell types can be specifically and rapidly ablated at any given time. In this proposal, we describe the generation and testing of a new method for increasing the specificity and ease of inducible cell ablation in mice. This system will contribute to modeling hearing loss and is adaptable for any cell type of interest to model other disorders resulting from cell degeneration.
| Quadros, Rolen M; Miura, Hiromi; Harms, Donald W et al. (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18:92 |
