DRG engraftment of transduced mesenchymal stem cells to treat neuropathic pain

描述(申请人提供)：神经性疼痛是常见的和致残的。尽管对疼痛机制的了解有所进步，但可用的治疗方法仍然不足。拟议研究的总体目标是通过结合两种新方法来设计一种高效和灵活的治疗系统。首先，针对单个(或多个)背根神经节(DRGs)的治疗将允许在节段性基础上直接治疗感觉神经元，从而限制对健康感觉通路的副作用。其次，间充质干细胞(MSCs)的移植为利用不断扩大的病理生理学理解提供了一个强大的机会。具体地说，自体MSCs很容易获得，很容易培养，经过基因工程后，它们具有高度的分泌肽的能力，这些细胞没有伦理或监管问题。初步数据表明，由于慢病毒载体将转基因插入宿主基因组中，并且MSCs分泌的治疗性多肽水平比未经基因修饰的MSCs高得多，因此在体外慢病毒转导在MSCs中经过多个繁殖周期后可以产生稳定的转基因表达。与直接将病毒载体注射到患者体内的基因治疗方法不同，该策略避免了病毒的免疫原性和毒性影响，并允许在重新植入之前在患者外部进行受控、高效的转导，从而提高了性能和安全性。这项拟议的工作将按顺序目标开发这种新疗法。首先，以大鼠为研究对象，研究骨髓间充质干细胞的必要剂量、存活率和分化命运。其次，重要的解剖学和生理学观察将评估MSC移植对内源性DRG细胞的影响，以证实MSCs本身的植入没有不良影响。最后，概念验证实验将在大鼠神经损伤模型中通过移植工程化的 MSCs进入背根节。这些翻译试验中使用的改良MSCs将分泌胶质细胞源性神经营养因子(GDNF)或白介素10，这两种细胞在通过其他途径输送时已证明在预防或逆转神经病理性疼痛方面有效。疼痛行为和细胞效应将与注射了只表达报告基因(GFP)的MSCs的动物进行比较，并与未经处理的对照组进行比较。拟议工作的完成将为工程间充质干细胞作为上述或其他止痛肽来源在较大动物或人类受试者中的治疗试验奠定基础。

DESCRIPTION (provided by applicant): Neuropathic pain is common and disabling. Although understanding of pain mechanisms has advanced, available treatments remain inadequate. The overall goal of the proposed research is to devise a highly effective and flexible treatment system by combining two novel approaches. First, therapy that targets a single (or several) dorsal root ganglia (DRGs) will permit direct treatment of sensory neurons on a segmental basis, thereby limiting side effects on healthy sensory pathways. Second, transplantation of mesenchymal stem cells (MSCs) provides a powerful opportunity to capitalize on expanding pathophysiological understanding. Specifically, autologous MSCs are readily available, they are easily grown, they are highly capable of secreting peptides after genetic engineering, and there are no ethical or regulatory issues with these cells. Preliminary data indicate that in vitro lentiviral transduction produces stable transgene expression in MSCs through many cycles of propagation, since this vector inserts the transgene into the host genome, and that MSCs secrete much higher levels of therapeutic peptides than they can without genetic modification. Unlike gene therapy approaches using direct viral vector injection into patients, this strategy avoids immunogenic and toxic effects of the virus, and permits controlled, highly efficient transduction outside the patient prior to reimplantation, thereby enhancing performance and safety. The proposed work will develop this new therapy in sequential Aims. First, the necessary dose of MSCs, their survival rates, and their differentiation fate will be characterized using rats as subjects. Second, important anatomical and physiological observations will evaluate effects of MSC transplantation on endogenous DRG cells to confirm that implantation of MSCs per se has no harmful effects. Finally, proof of concept experiments will measure the efficacy of treating neuropathic pain in a rat nerve-injury model, by transplantation of engineered MSCs into the DRG. The modified MSCs used in these translational trials will secrete either glial cell derived neurotrophic factor (GDNF) or interleukin 10, which have demonstrated efficacy in preventing or reversing neuropathic pain when delivered by other routes. Pain behavior and cellular effects will be compared to animals in which DRGs have been injected with MSCs that express only a reporter gene (GFP), and to untreated controls. Completion of the proposed work will establish the basis for therapeutic trials of engineered MSC as sources of these or other analgesic peptides in larger animals or human subjects.