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: 8228137
• 负责人: Stacey L. Halum
• 金额: $ 23.78万
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8228137
• 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; Engineering; Etiology; Experimental Designs; Functional disorder; Funding; Future; Gene Delivery; Gene Expression Microarray Analysis; Gene Transfer; 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人员，我感到很荣幸能够在医生和床边之间建立一座桥梁。我的长期职业目标是成为一名独立的临床医生-科学家调查员，为促进患者护理而开展最先进的NIH同行评议研究。具体地说，我想从事研究声带麻痹和其他喉部疾病的病理生理学和治疗选择的学术研究生涯。目前的应用包括通过自体肌肉干细胞(MSCs)喉部输送治疗物质(S)。虽然这一应用中的研究直接与声带麻痹的治疗有关，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的复发，为喉鳞状细胞癌提供辅助治疗，或扩大老年喉部的声带。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类临床试验，来自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，本提案中的实验被设计为纳入了不同的方法学。此外，为了帮助我的职业发展，我每周会与克拉普博士会面60分钟，讨论研究进展。我还将参加克拉普博士的研究小组并展示我的数据，从而加深我对各种电生理学、神经化学和分子生物学技术的理解(请参阅描述指导计划的支持函)。在此期间，我还期待与我的共同导师科内塔博士和伍德森博士进行定期讨论(见支持信)。最后，我将按照我的职业发展计划中的描述进行正式的课程学习。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等课程。生物信息学和生物统计学将对用于解决特定目标1的微阵列数据分析特别有帮助。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，这将为第三年开始的基因转移实验(特定目标3和4)做准备。 目前的职业发展(KO8)应用研究自体骨髓间充质干细胞治疗声带麻痹(VFP)的应用。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常导致发音困难并伴有声门性呼吸道阻塞。目前VFP的治疗方法并不理想，因为它们不能恢复动态运动。最近的研究表明，喉返神经损伤后持续的声带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发生的异常的、自发的再神经支配。这些实验的长期目标是使用临床上可行的技术来增强参与神经再生的生理通路到选定的喉肌，同时防止功能性拮抗神经再支配，从而潜在地恢复声带运动。具体地说，我们的目标是(1)利用微阵列和基因表达分析，以时间依赖的方式，确定与喉返神经再生相关的神经营养因子(NF)和神经营养因子受体(NFR)表达的定性和定量变化；(2)利用运动神经元培养和MSC存活试验，鉴定与RLN再生相关的神经营养因子，直接促进运动神经元生长和MSC存活；(3)构建编码治疗性神经营养因子的慢病毒载体，并在体外使慢病毒转导的原代肌肉干细胞最大限度地分泌神经营养因子。(4)利用喉返神经横断损伤的体内模型，在抑制功能性拮抗性外展运动的同时，通过自体MSC载体向喉内收肌治疗，从而潜在地恢复声带内收肌的运动。我们的初步研究表明，慢病毒载体可以有效地转导MSCs，并且分泌CNTF等神经营养因子的MSCs可以在失神经支配的半喉中存活至少两个月，这是一个足够的时间段，可以有效地促进神经再支配。基于大量自体骨髓间充质干细胞的容易获取和通过喉部注射的技术容易，该模型具有高度的临床适用性。事实上，当这个模型应用于人类时，手术将仅限于在办公室的局部麻醉剂下进行的小骨骼肌活组织检查，以及普通耳鼻喉科医生常规进行的喉部注射。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中出现的不良反应或排斥反应的风险。骨髓间充质干细胞是一种理想的基因传递干细胞，因为它们在培养中快速增殖，并具有防止肿瘤形成的先天功能。因此，该模型具有很高的可行性，并具有很大的治疗潜力。该模式在性质上也是假设产生的，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。