Neurotrophin-Secreting Muscle Stem Cell Therapy for Laryngeal Paralysis

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As a clinician and an investigator, I feel privileged to be in a key position to create a bridge between bench and bedside. My long-term career objective is to become an independent clinician-scientist investigator who conducts state-of-the-art NIH peer reviewed research for the advancement of patient care. Specifically, I would like to pursue an academic research career investigating the pathophysiology and therapeutic options for vocal fold paralysis and other laryngologic disorders. The current application involves laryngeal delivery of therapeutic substance(s) via autologous muscle stem cells (MSCs). While the studies in this application pertain directly to treatment of vocal fold paralysis, the model may ultimately be applied to other areas of laryngology such as controlling respiratory papillomatosis recurrence, providing adjuvant therapy for laryngeal squamous cell carcinoma, or augmenting vocal folds in presbylaryngis. As a clinician-scientist and laryngologist, I will be in a position to readily translate such basic science research into future human clinical trials, with funding from this grant being critical to her pursuit of such goals. To facilitate my goal of becoming a skilled, independent clinician-scientist, the experiments in this proposal have been designed to incorporate diverse methodologies. Additionally, to aid in my career development, I will meet with Dr. Clapp for 60 minutes each week to discuss research progress. I will also attend and present my data within Dr. Clapp's research group, thereby enhancing my understanding of a variety of electrophysiological, neurochemical and molecular biological techniques (see letter of support describing mentoring plan). During this time, I also anticipate having regular discussions with my co-mentors, Dr. Cornetta and Dr. Woodson (see letters of support). Finally, formal coursework will be taken as described in my Career Development Plan. In brief, I plan on taking courses in Molecular Biology Methodology, Biostatistics, Bioinformatics, and Experimental Design during year one. The Bioinformatics and Biostatistics will be especially helpful to the microarray data analysis used to address Specific Aim 1. During the second year of the award, I plan on taking an advanced course on Gene Transfer Approaches which will prepare me for the gene transfer experiments (Specific Aim 3 & 4) to be initiated in the third year. The current career development (KO8) application investigates therapeutic use of autologous MSCs for the treatment of vocal fold paralysis (VFP). VFP is a major etiology of communication disorders. While unilateral VFP can cause severe dysphonia and dysphagia, bilateral VFP often causes dysphonia with glottic airway obstruction. Current treatments for VFP are suboptimal in that they fail to restore dynamic motion. Recent studies suggest that persistent vocal fold immobility after recurrent laryngeal nerve (RLN) injury is not due to lack of reinnervation, but due to aberrant, spontaneous reinnervation which occurs after nearly all RLN injuries. The long-term goal of these experiments is to use clinically feasible techniques to enhance physiologic pathways involved in neural regeneration to selected laryngeal muscles while preventing functional antagonistic reinnervation, thereby potentially restoring vocal fold motion. Specifically, we aim (1) to use microarray and gene expression analysis in a time-dependent fashion after RLN injury to determine the qualitative and quantitative changes in neurotrophic factor (NF) and NF receptor (NFR) expression that are associated with RLN regeneration, (2) to use motoneuron culture and MSC survival assays to identify RLN- regeneration associated NFs that directly enhance motoneuron growth and MSC survival, (3) to construct a lentiviral vector encoding promising therapeutic NF, and maximize NF secretion in lentiviral transduced primary muscle stem cells in vitro, and (4) to use an in vivo model of RLN transection injury to therapeutically deliver NF via autologous MSC vectors to laryngeal adductor muscles after RLN injury while inhibiting functional antagonistic abduction, thereby potentially restoring vocal fold adductor motion. Our preliminary studies have demonstrated that MSCs can be efficiently transduced with lentiviral vector and that MSCs that secrete NF such as CNTF will survive in a denervated hemilarynx for at least a two month period, which is an adequate time period for NF delivery to effectively enhance reinnervation. The model is highly clinically applicable based on the ease of procurement of large quantities of autologous MSCs and the technical ease of delivery via laryngeal injection. In fact, when this model is applied to humans, surgeries would be limited to a small skeletal muscle biopsy which can be derived under local anesthetic in the office, and a laryngeal injection, which is a procedure routinely done by general otolaryngologists. The autologous nature of the cells also obviates risk of adverse reaction or rejection that is seen with synthetic material, cell lines, and allografts. MSCs are an ideal stem cell for gene delivery because they rapidly proliferate in culture and have innate features that protect against tumorigenesis. Thus, this model is highly feasible and holds great therapeutic potential for VFP. The model is also hypothesis-generating in nature, and will serve as a basis for future independent proposals, as is consistent with career development nature of the award.