Glycine Receptor Expression in Sensory Afferents to Modulate Pain Signaling

描述(由申请者提供)：慢性疼痛是一个主要的健康问题，影响着8000万美国人一生中的某个时候。目前的药物治疗并不是长期有效的，这导致了基因治疗方法的开发和测试。我们和其他人已经证明了基于单纯疱疹病毒1型(HSV)的载体可以在接种外周组织后将高效的痛觉调制转基因基因传递到体内的感觉神经元。这种方法的一个主要优点是，疼痛组织可以通过局部载体传递而被特定地靶向。我们认为，通过靶向负责慢性疼痛的特定神经元类型，从而使基因转移能够针对特定类型的疼痛，如炎症性或神经病理性疼痛，同时减少有害的副作用，这一优势可以进一步扩大。我们最近发现，单纯疱疹病毒介导的甘氨酸受体11亚单位(GlyR11)的转移和长期表达可以用来控制外源性甘氨酸注射的传入沉默的时间和持续时间。基于这些发现和我们最近在创建高效的、完全重定向的HSV载体方面的成功，我们假设我们可以选择性地沉默负责神经病理性和炎症性疼痛的不同的初级传入亚群，从而提供损伤特异性疼痛缓解。这些研究将使我们能够确定炎性和神经病理性疼痛的传入人群是相同的还是不同的，并为基于HSV的基因转移载体的未来发展提供合理的基础，该载体旨在限制相关初级传入的镇痛。我们预计，这些研究将(I)提供一种新的平台技术，使我们能够选择性地表达旨在调节感觉传入亚群功能的转基因，这一策略可以很容易地扩展到其他类型的感觉神经障碍；(Ii)开发感觉传入亚型的初始功能和物理图谱，这些亚型在沉默后将阻止不同的持续性疼痛状况，从而提供靶向疼痛控制所需的必要信息；(Iii)识别已功能改变的传入以对疼痛刺激做出反应，提供关于神经纤维可塑性的进一步信息；以及，(Iv)确定与使不适当的感觉神经元群体沉默相关的潜在风险(例如，本体感觉改变)。为了实现这些目标，我们提出了一系列相互关联的实验，描述了三个具体的目标。在目标1中，HSV载体的感染性将被重定向以选择性地转导(A)A2纤维，(B)肽能和(C)非肽能C纤维。在目标2中，我们将使用转基因启动子来补充转导重定向和转录靶向，当与转导靶向结合时，将微调沉默特异性。这一组合策略旨在创建更大的传导靶向群体中感觉纤维亚群的初始精细图谱，这些群体代表对不同痛苦刺激的反应的关键传入。在目标3中，通过周围接种炎症和神经病理性疼痛的动物模型，将重定向HSV载体导入DRG，并建立甘氨酸诱导的GlyR11介导的沉默后的止痛效果和副作用。 公共卫生相关性：慢性疼痛是一个主要的健康问题，影响着8000万美国人一生中的某个时候。目前的治疗方法不是长期有效的，造成了大量未得到满足的医疗需求。本研究中描述的实验旨在深入了解特定伤害性感受器群体在慢性疼痛反应中的作用，这可能导致通过靶向传递高度特异调控的疼痛沉默基因疗法来控制疼痛的方法。

DESCRIPTION (provided by applicant): Chronic pain is a major health concern affecting 80 million Americans at some time in their lives. Current pharmacotherapies are not effective long-term, which has led to the development and testing of gene therapy approaches. We and others have demonstrated that herpes simplex virus type 1 (HSV) based vectors can deliver highly effective pain-modulating transgenes to sensory neurons in vivo following inoculation of peripheral tissue. One major advantage of this approach is that painful tissue can be specifically targeted by local vector delivery. We believe that this advantage can be further extended by targeting specific neuron types responsible for chronic pain, thus enabling gene transfer to be tailored to specific types of pain, such as inflammatory or neuropathic pain, while simultaneously reducing deleterious side effects. We have recently shown that HSV-mediated transfer and long-term expression of the glycine receptor 11 subunit (GlyR11) can be used to control the timing and duration of afferent silencing with exogenous administration of glycine. Based on these findings and our recent success in the creation of highly efficient, fully retargeted HSV vectors, we hypothesize that we can selectively silence distinct subpopulations of primary afferents responsible for neuropathic and inflammatory pain, therefore providing injury specific pain relief. These studies will enable us to determine whether the same or different afferent populations underlie inflammatory and neuropathic pain and provide the rational basis for the future development of HSV-based gene transfer vectors designed to restrict analgesia to the relevant primary afferents. We anticipate that these studies will (i) provide a novel platform technology that will allow us to selectively express transgenes designed to modulate the function of sensory afferent subpopulations, a strategy that can be readily extended to other types of sensory nerve disorders; (ii) develop initial functional and physical maps of sensory afferent subtypes that upon silencing will block different persistent pain conditions, thereby providing essential information needed for targeted pain control; (iii) identify afferents that have been functionally altered to respond to painful stimuli providing further information on nerve fiber plasticity; and, (iv) identify potential risks associated with silencing of an inappropriate population of sensory neurons (e.g. altered proprioception). To achieve these goals, we have proposed a series of interrelated experiments described in 3 Specific Aims. In Aim 1, the infectivity of HSV vectors will be retargeted to selectively transduce (a) A2 fibers, (b) peptidergic and (c) nonpeptidergic C fibers. In Aim 2, we will complement transductional retargeting with transcriptional targeting using transgene promoters that will, when combined with transductional targeting, fine tune silencing specificity. The combination strategy is intended to create initial fine maps of subpopulations of sensory fibers within the larger transductionally targeted groups representing critical afferents for the response to different painful stimuli. In Aim 3, the retargeted HSV vectors will be introduced into the DRG by peripheral inoculation of animal models of inflammatory and neuropathic pain and the analgesic efficacy and side effect profiles will be established following glycine-induced GlyR11-mediated silencing. PUBLIC HEALTH RELEVANCE: Chronic pain is a major health concern affecting 80 million Americans at some time in their lives. Current treatments are not effective long-term, creating a substantial unmet medical need. Experiments described in this study are intended to develop insights into the role of specific nociceptor populations in chronic pain responses that may lead to methods to control pain by targeted delivery of a highly specific regulated pain silencing gene therapy.