Neurotrophin-Secreting Muscle Stem Cell Therapy for Laryngeal Paralysis

神经营养蛋白分泌肌干细胞治疗喉麻痹

• 批准号: 8431796
• 负责人: Stacey L. Halum
• 金额: $ 23.64万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431796
• 关键词: Address; Adjuvant Therapy; Adverse reactions; Allografting; Animal Model; Animals; Area; Atrophic; Autologous; Award; Basic Science; Bilateral; Bioinformatics; Biological; Biological Assay; Biometry; Biopsy; Cell Line; Cell Survival; Cells; Ciliary Neurotrophic Factor; Clinical; Clinical Trials; Communication impairment; Data; Data Analyses; Deglutition; Deglutition Disorders; Development Plans; Disease; Dysphonia; Etiology; Experimental Designs; Functional disorder; Funding; Future; Gene Delivery; Gene Expression Microarray Analysis; Gene Transfer; Genetic Engineering; Goals; Grant; Growth; Human; In Vitro; Injection of therapeutic agent; Injury; Laryngeal Paralysis; Laryngeal Squamous Cell Carcinoma; Laryngeal muscle structure; Larynx; Lentivirus Vector; Letters; Local Anesthetics; Mentors; Methodology; Methods; Microarray Analysis; Modeling; Molecular; Molecular Biology; Motion; Motor Neurons; Muscle; Muscle satellite cell; Natural regeneration; Nature; Nerve Growth Factor Receptors; Nerve Growth Factors; Nerve Regeneration; Operative Surgical Procedures; Otolaryngologist; Paralysed; Pathway interactions; Patient Care; Patients; Physiological; Positioning Attribute; Procedures; Proliferating; Recurrence; Recurrent Laryngeal Nerve; Research; Research Peer Review; Research Personnel; Risk; Scientist; Skeletal Muscle; Stem cells; Synkinesis; Techniques; Therapeutic; Therapeutic Uses; Time; Translating; United States National Institutes of Health; Voice; airway obstruction; attenuation; base; bench to bedside; career; career development; clinical application; design; in vivo Model; meetings; nerve injury; neurochemistry; neurotrophic factor; prevent; receptor expression; reinnervation; research study; respiratory; restoration; stem cell therapy; tumorigenesis; vector; vocal cord

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.

作为一名临床医生和研究者，我很荣幸能够在一个关键的位置上建立一个连接工作台和床边的桥梁。我的长期职业目标是成为一名独立的临床科学家调查员，进行最先进的NIH同行评审研究，以促进患者护理。具体来说，我想从事学术研究事业，调查声带麻痹和其他喉部疾病的病理生理学和治疗方案。本申请涉及通过自体肌肉干细胞（MSC）喉部递送治疗物质。虽然本申请中的研究直接涉及声带麻痹的治疗，但该模型最终可应用于喉学的其他领域，例如控制呼吸性乳头状瘤病复发，为喉鳞状细胞癌提供辅助治疗，或增强老年性喉的声带。作为一名临床科学家和喉部专家，我将能够随时将这些基础科学研究转化为未来的人类临床试验，这笔赠款的资金对她追求这些目标至关重要。 为了促进我成为一名熟练的，独立的临床科学家的目标，本提案中的实验旨在纳入不同的方法。此外，为了帮助我的职业发展，我每周都会与Clapp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，从而提高我对各种电生理学、神经化学和分子生物学技术的理解（见描述指导计划的支持信）。在此期间，我也期待与我的共同导师，博士科内塔和博士伍德森定期讨论（见支持信）。最后，正式的课程将采取在我的职业发展计划中描述。简而言之，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。在获奖的第二年，我计划参加一个关于基因转移方法的高级课程，这将为我准备在第三年启动的基因转移实验（具体目标3和4）。 目前的职业发展（KO8）应用研究了自体MSC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可导致严重的发音困难和吞咽困难，而双侧VFP则常导致发音困难伴声门型气道阻塞。VFP的当前治疗是次优的，因为它们不能恢复动态运动。最近的研究表明，喉返神经（RLN）损伤后持续性声带不动不是由于缺乏神经再支配，而是由于异常，自发性神经再支配，几乎所有的RLN损伤后发生。这些实验的长期目标是使用临床上可行的技术，以增强参与神经再生的生理途径，同时防止功能性拮抗神经再支配，从而可能恢复声带运动选择喉肌。具体地，我们的目标是（1）在RLN损伤后以时间依赖性方式使用微阵列和基因表达分析，以确定与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存活测定来鉴定直接增强运动神经元生长和MSC存活的RLN再生相关NF，（3）构建编码有前景的治疗性NF的慢病毒载体，并在体外使慢病毒转导的原代肌肉干细胞中的NF分泌最大化，和（4）使用RLN横断损伤的体内模型，在RLN损伤后通过自体MSC载体将NF治疗性递送至喉内收肌，同时抑制功能性拮抗性外展，从而潜在地恢复声带内收肌运动。我们的初步研究已经证明，MSC可以有效地转导慢病毒载体，并分泌NF如CNTF的MSC将在去神经支配的半喉中存活至少两个月，这是NF递送有效增强神经再支配的足够时间。该模型是高度临床适用的基础上，容易采购大量的自体骨髓间充质干细胞和技术容易通过喉注射输送。事实上，当这种模型应用于人类时，手术将仅限于在办公室局部麻醉下进行的小骨骼肌活检和喉注射，这是一般耳鼻喉科医生常规进行的手术。细胞的自体性质也避免了合成材料、细胞系和同种异体移植物所见的不良反应或排斥反应的风险。间充质干细胞是基因递送的理想干细胞，因为它们在培养中迅速增殖，并具有防止肿瘤发生的先天特征。因此，该模型是高度可行的，并且对VFP具有巨大的治疗潜力。该模式也是假设生成的性质，并将作为未来独立提案的基础，因为这符合该奖项的职业发展性质。