Combining new gene therapy with non-invasive spinal roots stimulation to improve synaptic plasticity at spino-muscular circuitry after spinal cord injury

自愿行走能力受损和膀胱功能障碍是退伍军人中的一个严重问题， 脊髓损伤（SCI）。最近的研究结果，包括我们自己的，显示至少有三个 已知限制SCI恢复的主要因素：（1）神经元兴奋性降低，（2）轴突的存在 生长/再生抑制剂，和（3）缺乏神经营养因子支持。利用动物模型，我们最近 证明了瘢痕相关的抑制性硫酸软骨素蛋白聚糖（CSPG）的降解， 软骨素酶-ABC（ChABC）与基于AAV的神经营养因子NT 3递送组合，诱导了 轻度挫伤SCI后部分改善。使用ChABC的一个潜在缺点是， 非特异性的，即降解所有CSPG，包括作为细胞外基质的重要组分的那些。 在我们寻找更特异性的靶点时，我们最近证明了一种CSPG分子，NG 2， 被认为是脑和脊髓损伤后轴突再生的主要障碍， 传导，但其他CSPG测试没有。急性给予单克隆NG 2功能中和 抗体（NG 2-Ab;最初设计用于防止NG 2对轴突生长的抑制作用）阻止了 急性注射NG 2到脊髓中引起的传导阻滞。鞘内输注NG 2-Ab，通过 然而，渗透压微型泵2周仅引起有限和短暂的运动功能改善 SCI之后。为了设计一种安全、延长和临床上可行的NG 2- 抗体，我们已经成功地创建了一种新的基于AAV-10载体的基因治疗工具，用于长期和临床- 重组单链可变片段（scFv）抗NG 2抗体：AAV-NG 2Ab的相关递送。 初步实验的结果显示，AAV-NG 2Ab和AAV-NT 3的组合施用可显著地降低AAV-NG 2Ab和AAV-NT 3的表达。 与ChABC/AAV-NT 3相比，在轻度（150 kDyn）挫伤后，影响 然而，这种新的基因治疗工具（AAV-NG 2Ab/AAV-NT 3）对大鼠运动恢复的作用仍然有限 而重度挫伤组损伤程度较轻。 为了进一步改善AAV-NG 2Ab/AAV-NT 3的有益效果并扩大对AAV-NG 2Ab/AAV-NT 3的改善， 严重SCI模型，我们现在建议增加第三种治疗成分，即非侵入性重复电 脊柱磁刺激（rSEMS）。我们最近发现rSEMS加强了传输 并改善运动神经元突触输入处的NMDA受体的功能，这是启动 NT-3在这些输入。因此，在建议的项目中，我们设计了一种新的添加剂处理， AAV 10-NG 2 Ab、AAV 10-NT 3和rSEMS。除了轻度挫伤损伤模型外，我们建议使用 已知可引起运动功能和膀胱活动严重缺陷的严重胸中部挫伤 在大鼠和人SCI中。这项研究的一个重要和新颖的方面是评估提出的新基因 治疗（AAV-NG 2Ab/AAV-NT 3）联合rSEMS对膀胱功能的影响。为了评估这些药物的疗效， 治疗，我们将进行多学科的检查，包括在体内生理学，解剖学， 免疫化学和行为。我们将研究新的治疗方法的效果（累加或协同）。 （1）加强通过损伤震中至腰运动神经元的突触连接，和 然后到后肢肌肉（使用体内电生理学）;（2）纤维的解剖可塑性， 暴露于这种新的治疗后突触反应的持续性（使用解剖追踪和 共聚焦显微镜）;（3）运动性能的恢复（使用自动化Catwalk步态分析）;（4） 恢复膀胱活动（使用代谢室和膀胱测压/电生理学）。更好地了解 在细胞水平的治疗效果，我们将研究轴突髓鞘再生（使用电子显微镜）和NG 2- 接触兰维尔结的阳性突起。正在进行的实验的初步结果表明， 在严重胸中部挫伤性SCI后接受这种新型添加剂治疗的大鼠的运动功能。

Impaired ability of voluntary walking and bladder dysfunction is an acute problem among veterans with spinal cord injuries (SCI). Results of recent studies, including our own, revealed that there are at least three major factors known to limit recovery from SCI: (1) decreased neuronal excitability, (2) presence of axonal growth/regeneration inhibitors, and (3) lack of neurotrophin support. Using animal models, we have recently demonstrated that degradation of scar-related inhibitory Chondroitin Sulfate Proteoglycans (CSPGs) with the enzyme Chondroitinase-ABC (ChABC), combined with AAV-based delivery of neurotrophin NT3, induced partial improvements following mild contusion SCI. A potential disadvantage to the use of ChABC is that it is not specific, i.e. degrades all CSPGs, including those that are important components of the extracellular matrix. In our search for more specific targets, we have recently demonstrated that one CSPG molecule, NG2, known as a major obstacle to axonal regeneration following brain and spinal cord injury, blocks axonal conduction, but other CSPGs tested did not. Acute administration of monoclonal NG2 function neutralizing antibody (NG2-Ab; designed initially to prevent inhibitory effects of NG2 on axonal growth) prevents the conduction block induced by acute injections of NG2 into the spinal cord. Intrathecal infusion of NG2-Ab, via osmotic mini-pump for 2 weeks, however, induced only limited and transient improvements of motor function following SCI. In an attempt to design an approach for safe, prolonged and clinically feasible delivery of NG2- Ab, we have successfully created a new AAV-10 vector-based gene therapy tool for prolonged and clinically- relevant delivery of a recombinant single chain variable fragment (scFv) anti-NG2 antibody: AAV-NG2Ab. Results of preliminary experiments revealed that combined administration of AAV-NG2Ab and AAV-NT3 induced greater improvements, compared to ChABC/AAV-NT3, following mild (150 kDyn) contusions. Effects of this novel gene therapy (AAV-NG2Ab/AAV-NT3) tool on motor recovery were, however, still limited in rats with mild contusion and less obvious in rats with severe contusion SCI. In attempts to further improve the beneficial effects of AAV-NG2Ab/AAV-NT3 and expand improvements to severe SCI models, we now propose to add a third treatment component, i.e. non-invasive repetitive electro- magnetic stimulation over spinal vertebrae (rSEMS). We recently found that rSEMS strengthens transmission and improves function of NMDA receptor at motoneuron synaptic inputs, which is required to initiate effects of NT-3 at these inputs. Thus, in the proposed project we have designed a new additive treatment comprised of AAV10-NG2Ab, AAV10-NT3 and rSEMS. In addition to a mild contusion model of injury, we propose to use severe mid-thoracic contusions which are known to induce major deficits of motor function and bladder activity in rat and human SCI. An important and novel aspect of this research is evaluation of the proposed novel gene therapy (AAV-NG2Ab/AAV-NT3) combined with rSEMS on bladder function. To evaluate the efficacy of these treatments, we will conduct a multidisciplinary examination, including in vivo physiology, anatomy, immunochemistry and behavior. We will examine the effects (additive or synergistic) of the new therapeutic treatment on (1) strengthening synaptic connections through the injury epicenter to lumbar motoneurons, and then to hindlimb muscles (using in-vivo electrophysiology); (2) anatomical plasticity of fibers accounting for the persistence of the synaptic response after exposure to this novel treatment (using anatomical tracing and confocal microscopy); (3) recovery of locomotor performance (using automated Catwalk gait analyses); (4) recovery of bladder activity (using metabolic chamber and cystometry/ electrophysiology). To better understand the effects of treatment at the cellular level, we will study axon remyelination (using Electron Microscopy) and NG2- positive processes contacting nodes of Ranvier. Preliminary results of on-going experiments show improvements of motor function in rats that have received this novel additive treatment after severe mid-thoracic contusive SCI.