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: 8610282
• 负责人: Stacey L. Halum
• 金额: $ 22.94万
• 依托单位: PURDUE UNIVERSITY
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8610282
• 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.

作为临床医生和调查员，我感到有荣幸能够在长凳和床边建立桥梁的关键位置。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，他进行了最先进的NIH同伴审查了对患者护理的发展研究。具体来说，我想从事一项学术研究职业，研究声带瘫痪和其他喉咙疾病的病理生理学和治疗选择。当前的应用涉及喉部通过自体肌肉干细胞（MSC）递送治疗物质。虽然该应用中的研究直​​接与声带瘫痪有关，但该模型最终可用于喉部的其他领域，例如控制呼吸乳头状瘤病复发，为喉部的鼻鳞状细胞癌提供辅助治疗，或增强老年癌中的声音褶皱。作为临床医生科学家和喉科医生，我将可以轻松地将这种基础科学研究转化为未来的人类临床试验，而这笔赠款的资金对于她追求此类目标至关重要。 为了促进我成为一个熟练的独立临床医生科学家的目标，该提案中的实验旨在结合多种方法。此外，为了帮助我的职业发展，我每周将与Clapp博士见面60分钟，讨论研究进度。我还将在Clapp博士的研究小组中参加并介绍我的数据，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（请参阅描述指导计划的支持信）。在这段时间里，我还希望与我的院长Cornetta博士和Woodson博士进行定期讨论（请参阅支持信）。最后，将按照我的职业发展计划中的描述进行正式课程。简而言之，我计划参加第一年的分子生物学方法论，生物统计学，生物信息学和实验设计的课程。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用。在奖励的第二年，我计划在第三年开始对基因转移实验（特定目标3和4）的基因转移方法的高级课程，这将为我做好准备。 当前的职业发展（KO8）申请研究了自体MSC治疗声带瘫痪（VFP）的治疗用途。 VFP是沟通障碍的主要病因。虽然单方面的VFP会引起严重的烦躁不安和吞咽困难，但双侧VFP通常会引起吞咽困难，并带有震颤的气道阻塞。 VFP的当前治疗方法是次优的，因为它们无法恢复动态运动。最近的研究表明，复发性喉神经（RLN）损伤后的持续性声折叠不动，而是由于缺乏加剧而导致的，而是由于几乎所有RLN损伤后发生的异常，自发的加剧。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途径，同时预防功能拮抗作用的重新支配，从而潜在地恢复人声折叠运动。具体而言，我们的目的是（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，以确定神经营养因子（NF）和NF受体（NFR）表达的定性和定量变化，与RLN再生相关，（2）使用Motonerron培养和MSSc Intedn dike Interip n. Remcorsion rln-remons inf inf inf Regiated rln-remons inf inf inf inf inf in。生存期，（3）构建编码有前途的治疗NF的生病病毒载体，并最大化慢病毒转导的NF分泌，可在体外进行过转导的原发性肌肉干细胞，（4）使用RLN过渡损伤的体内模型，以通过治疗的NF造成NF造成NF的自动性MSC Vectors to larary Mussc，从绑架，从而有可能恢复声带内收肌运动。我们的初步研究表明，MSC可以通过慢病毒载体有效地转导，并且分泌NF（例如CNTF）的MSC将在未经new的Hemilarynx中生存至少两个月，这是NF的足够时间段，可以有效地增强NF的递送。该模型在临床上是基于易于采购的大量自体MSC和通过喉部注射的技术易交付性的高度适用。实际上，当该模型应用于人类时，手术将仅限于可以在办公室中局部麻醉下衍生的小骨骼肌活检，而喉注注射则是普通耳鼻喉科医生通常进行的。细胞的自体性也消除了与合成物质，细胞系和同种异体移植物看到的不良反应或排斥的风险。 MSC是用于基因递送的理想干细胞，因为它们在培养中迅速增殖，并且具有可预防肿瘤发生的先天特征。因此，该模型是高度可行的，并且具有VFP的巨大治疗潜力。该模型本质上也是假设生成的，并且将作为未来独立建议的基础，这与奖项的职业发展性质一致。

项目成果

Neurotrophic factor-secreting autologous muscle stem cell therapy for the treatment of laryngeal denervation injury.

• DOI: 10.1002/lary.23519
• 发表时间: 2012-11
• 期刊: LARYNGOSCOPE
• 影响因子: 2.6
• 作者: Halum, Stacey L.;McRae, Bryan;Bijangi-Vishehsaraei, Khadijeh;Hiatt, Kelly
• 通讯作者: Hiatt, Kelly

A rapid, novel model of culturing cranial nerve X-derived motoneurons for screening trophic factor outgrowth response.

一种快速、新颖的培养脑神经 X 源运动神经元的模型，用于筛选营养因子生长反应。

• DOI: 10.1179/1743132812y.0000000046
• 发表时间: 2012
• 期刊: Neurological research
• 影响因子: 1.9
• 作者: McRae,BryanR;Shew,Matthew;Aaron,GeoffreyP;Bijangi-Vishehsaraei,Khadijeh;Halum,StaceyL
• 通讯作者: Halum,StaceyL