A long term goal of our laboratory is to define gene-based strategies that restore auditory and vestibular function in the diseased or damaged inner ear. Significant barriers to progress in the field of regenerative medicine are the identification f genes that have authentic therapeutic potential and the creation of reliable strategies to efficaciously modulate their expression and function. To begin to address these barriers, we have devised in utero gene transfers techniques that permit gain-of-function studies in the developing mouse inner ear that rely on viral vectors and in vivo electroporation. In the present proposal, we seek to define a rapid, cost effective, and technically simplified experimental paradigm that enables modulation of gene expression in otic precursors by in vivo protein transduction. Virtually all proteins do not spontaneously enter cells which restricts their usefulness as research tools. However, two new technologies have emerged that show enormous potential: surface remodeling of proteins and virus-like particles. Surface remodeling of proteins by replacement of nonconserved residues facilitates endocytosis in part by maximizing productive interactions with sulfated proteoglycans in the glycocalyx. Next generation virus-like particles are derived from an avian viral vector and effectively deliver a protein rather than a nucleic acid payload to the infected cell.
In Aim 1, we propose to initiate somatic recombination in otic precursors by transuterine microinjection of bioactive Cre recombinase using the surface remodeling and virus-like particle formats. In subaim A, we test both formats using a floxed allele of a fluorescent reporter to define the time course of recombination, the type and distribution of recombined cells, and the potential impact of these reagents on postnatal acquisition of hearing and balance. In subaim B, we will generate inner ears mosaic for atonal homolog 1 (Atoh1) expression by Cre-mediated recombination of the floxed Atoh1 gene. We predict that abrogation of Atoh1 expression will reduce the number of sensory hair cells formed and allow us to test the hypothesis that Atoh1 positive cells can instruct the formation of Atoh1 negative hair cells. An additional property of surface remodeled proteins is their ability to reversibly complex with nucleic acids while retaining their protein transduction characteristics.
In Aim 2, we propose to transfect otic precursors with expression plasmid or small interfering RNA (siRNA) by transuterine microinjection of surface remodeled protein/nucleic acid complexes. In subaim A, we will define the parameters for efficient expression plasmid transfection and test the bioactivity of an Atoh1 construct which is predicted to induce the formation of extra hair cells. In subaim B, we will define the parameters for efficiet siRNA transfection and test the bioactivity of siRNAs directed against Atoh1 to knock down gene expression and perturb hair cell fate specification. Successful completion of the proposed studies will establish a gain- and loss-of-function experimental platform to discern genes that have therapeutic potential and will introduce in vivo protein transduction as a potential therapeutic strategy for regenerative interventions in the diseased inner ear.
This proposal will test the ability of a novel class of protein-based reagents to reliably modulate gene expression in the developing mouse inner ear in utero. The ability to turn on and turn off gene expression at defined time points in the developing sensory and non sensory regions of the mammalian inner ear will advance knowledge of how gene expression is regulated. This new knowledge can be exploited to define and test gene-based therapies to restore auditory and vestibular function in mutant mice that model human inner ear disease.
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|Huang, Alice H; Riordan, Timothy J; Wang, Lingyan et al. (2013) Repositioning forelimb superficialis muscles: tendon attachment and muscle activity enable active relocation of functional myofibers. Dev Cell 26:544-51|