Glycine Receptor Expression in Sensory Afferents to Modulate Pain Signaling

描述（由申请人提供）：慢性疼痛是影响8000万美国人一生中某个时期的主要健康问题。目前的药物治疗不是长期有效的，这导致了基因治疗方法的发展和测试。我们和其他人已经证明，基于1型单纯疱疹病毒（HSV）的载体在接种外周组织后，可以在体内向感觉神经元传递高效的疼痛调节转基因。这种方法的一个主要优点是疼痛组织可以通过局部载体递送特异性靶向。我们相信，这一优势可以通过针对负责慢性疼痛的特定神经元类型进一步扩展，从而使基因转移能够针对特定类型的疼痛，如炎症性或神经性疼痛，同时减少有害的副作用。我们最近的研究表明，单纯疱疹病毒介导的甘氨酸受体11亚基（GlyR11）的转移和长期表达可用于控制外源性甘氨酸传入沉默的时间和持续时间。基于这些发现和我们最近在高效、完全重定向的HSV载体创造方面的成功，我们假设我们可以选择性地沉默负责神经性和炎症性疼痛的原发性传入事件的不同亚群，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症性和神经性疼痛的传入神经群是否相同或不同，并为未来开发基于单纯疱疹病毒的基因转移载体提供合理的基础，旨在将镇痛限制在相关的初级传入神经。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，这种策略可以很容易地扩展到其他类型的感觉神经疾病；（ii）开发感觉传入亚型的初始功能和物理图谱，在沉默后将阻断不同的持续性疼痛状况，从而为有针对性的疼痛控制提供必要的信息；（iii）识别为响应疼痛刺激而发生功能改变的传入神经，为神经纤维可塑性提供进一步的信息；（iv）识别与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关的实验，详见3 Specific Aims。在Aim 1中，HSV载体的传染性将被重新定位，以选择性地转导(a) A2纤维，(b)多肽能纤维和(c)非多肽能c纤维。在Aim 2中，我们将使用转基因启动子补充转导重靶向和转录靶向，当与转导靶向结合时，这些启动子将微调沉默特异性。该组合策略的目的是在较大的转导目标群中创建感觉纤维亚群的初始精细图，这些群体代表对不同疼痛刺激的反应的关键传入。在Aim 3中，通过外周接种炎症性和神经性疼痛动物模型，将重靶向HSV载体引入DRG，并在甘氨酸诱导的glyr11介导的沉默后建立镇痛效果和副作用概况。

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.