Neurotrophin-Secreting Muscle Stem Cell Therapy for Laryngeal Paralysis

描述（由申请人提供）：作为一名临床医生和研究者，我很荣幸能够在临床和临床之间建立桥梁。我的长期职业目标是成为一名独立的临床医生兼科学家调查员，为提高患者护理水平进行最先进的NIH同行评议研究。具体来说，我想从事一项学术研究，研究声带麻痹和其他喉部疾病的病理生理学和治疗方案。目前的应用包括通过自体肌肉干细胞（MSCs）喉部递送治疗物质。虽然本应用的研究直接涉及到声带麻痹的治疗，但该模型最终可能应用于喉科的其他领域，如控制呼吸道乳头状瘤病复发、喉部鳞状细胞癌的辅助治疗、喉部老年性声带增强等。作为一名临床科学家和喉科医生，我将能够很容易地将这些基础科学研究转化为未来的人体临床试验，这笔资助对她实现这些目标至关重要。为了促进我成为一名熟练的、独立的临床科学家的目标，本提案中的实验被设计成包含多种方法。此外，为了帮助我的职业发展，我将每周与克拉普博士会面60分钟，讨论研究进展。我还将参加Clapp博士的研究小组并展示我的数据，从而增强我对各种电生理，神经化学和分子生物学技术的理解（见支持信描述指导计划）。在这段时间里，我也期待与我的共同导师Cornetta博士和Woodson博士（见支持信）进行定期讨论。最后，正式的课程将按照我的职业发展计划进行。总之，我计划在第一年学习分子生物学方法论、生物统计学、生物信息学和实验设计。生物信息学和生物统计学将特别有助于用于解决特定目标1的微阵列数据分析。在获得奖项的第二年，我计划学习一门关于基因转移方法的高级课程，这将为我在第三年开始的基因转移实验（Specific Aim 3 & 4）做准备。目前的职业发展（KO8）应用程序研究了自体MSCs治疗声带麻痹（VFP）的治疗用途。VFP是沟通障碍的主要病因。单侧VFP可引起严重的发声障碍和吞咽困难，而双侧VFP常引起声门气道阻塞的发声障碍。目前对VFP的治疗是次优的，因为它们不能恢复动态运动。最近的研究表明，喉返神经损伤后声带持续不动不是由于缺乏再神经支配，而是由于几乎所有喉返神经损伤后出现的异常、自发的再神经支配。这些实验的长期目标是使用临床可行的技术来增强参与神经再生的生理途径，以选择喉部肌肉，同时防止功能性拮抗神经再支配，从而有可能恢复声带运动。具体来说，我们的目标是(1)在RLN损伤后以时间依赖的方式使用微阵列和基因表达分析来确定与RLN再生相关的神经营养因子（NF）和NF受体（NFR）表达的定性和定量变化，(2)使用运动神经元培养和MSC存活测定来识别与RLN再生相关的NF，这些NF直接增强运动神经元的生长和MSC存活。(3)构建一个慢病毒载体，编码有治疗前景的NF，并在体外使慢病毒转导的原代肌肉干细胞中最大限度地分泌NF。(4)利用RLN横断损伤的体内模型，在RLN损伤后通过自体间充质干细胞载体将NF治疗性地输送到喉内收肌，同时抑制功能性拮抗外展，从而有可能恢复声带内收肌的运动。我们的初步研究表明，MSCs可以被慢病毒载体有效地转导，并且分泌NF（如CNTF）的MSCs可以在去神经的半喉部存活至少两个月，这是一个足够的时间来传递NF以有效地增强神经再生。该模型易于获得大量的自体间充质干细胞，并且易于通过喉部注射给药，因此具有很高的临床适用性。事实上，当这个模型应用于人类时，手术将局限于一个小的骨骼肌活检可以在办公室的局部麻醉下进行，以及喉注射，这是普通耳鼻喉科医生的常规手术。细胞的自体特性也避免了合成材料、细胞系和同种异体移植物出现不良反应或排斥反应的风险。间充质干细胞是一种理想的基因传递干细胞，因为它们在培养中增殖迅速，并且具有防止肿瘤发生的先天特征。因此，该模型是高度可行的，具有很大的治疗VFP的潜力。该模型在本质上也是产生假设，并将作为未来独立提案的基础，这与该奖项的职业发展性质是一致的。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Vocal fold (voice box) paralysis is a major cause of communication disorders, causing many patients to suffer from severe voice, swallowing and airway problems. The goal of these experiments is to use muscle stem cells genetically engineered to secrete nerve growth factors to treat vocal fold paralysis, potentially having great clinical implications for patients suffering from this disorder and other related diseases.