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

将新基因疗法与非侵入性脊髓根刺激相结合，以改善脊髓损伤后脊髓肌肉回路的突触可塑性

• 批准号: 10158426
• 负责人: Victor L Arvanian
• 金额: --
• 依托单位: NORTHPORT VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158426
• 关键词: Accounting; Action Potentials; Acute; Adult; Afghanistan; Aftercare; Anatomy; Animal Model; Antibodies; Axon; Behavior; Bladder; Bladder Dysfunction; CSPG4 gene; Caring; Chest; Chondroitin ABC Lyase; Chondroitin Sulfate Proteoglycan; Chronic; Cicatrix; Clinic; Clinical; Clinical Trials; Collaborations; Confocal Microscopy; Contusions; Corticospinal Tracts; Development; Disadvantaged; Disease; Dorsal; Electric Stimulation; Electromagnetics; Electron Microscopy; Electrophysiology (science); Enzymes; Evaluation; Exposure to; Extracellular Matrix; Fiber; Funding; Gait; Genes; Glutamate Receptor; Goals; Growth; Hair; Hindlimb; Human; Immunochemistry; Impairment; Individual; Infusion procedures; Injections; Injury; Lateral; Leg; Locomotor Recovery; Magnetism; Measures; Mediating; Membrane; Metabolic; Military Personnel; Modeling; Motor; Motor Neurons; Muscle; N-Methyl-D-Aspartate Receptors; NTF3 gene; Natural regeneration; Neurogenic Bladder; Neurons; Oligodendroglia; Output; Pathway interactions; Pattern; Performance; Physiological; Physiology; Process; Property; Ranvier' s Nodes; Rattus; Recombinants; Recovery; Reflex action; Research; Residual state; Resistance; Spinal; Spinal Cord; Spinal Cord Contusions; Spinal Injections; Spinal cord damage; Spinal cord injury; Spinal nerve root structure; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Treatment Efficacy; Urethral sphincter; Urination; Urine; Veterans; Viral Vector; Walking; War; Withdrawal; anatomical tracing; axon growth; axon regeneration; base; blood-brain barrier function; clinically relevant; density; design; effective therapy; efficacy evaluation; experimental study; gait examination; gene therapy; improved; improved functioning; in vivo; inhibitor/antagonist; motor deficit; motor function improvement; motor function recovery; motor recovery; multidisciplinary; nerve supply; neural circuit; neuronal excitability; neurotrophic factor; neutralizing antibody; novel; novel therapeutics; oligodendrocyte progenitor; osmotic minipump; pressure; prevent; remyelination; response; spinal cord and brain injury; spine bone structure; stem cells; synaptic function; tool; transgene delivery; transmission process; treatment effect; vector; white matter

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.

自主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，包括我们自己的研究，表明至少有三个 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，以及（3）缺乏神经营养蛋白的支持。最近，我们利用动物模型 证明疤痕相关的抑制性硫酸软骨素蛋白多糖（CSPG）的降解与 软骨素酶-ABC (ChABC) 与基于 AAV 的神经营养素 NT3 递送相结合，诱导 轻度挫伤 SCI 后部分改善。使用 ChABC 的一个潜在缺点是 不具有特异性，即降解所有 CSPG，包括那些细胞外基质的重要组成部分。 在寻找更具体的靶标时，我们最近证明了一种 CSPG 分子 NG2， 被认为是大脑和脊髓损伤后轴突再生的主要障碍，阻碍轴突 传导，但其他测试的 CSPG 却没有。单克隆NG2功能中和的急性给药 抗体（NG2-Ab；最初设计用于防止 NG2 对轴突生长的抑制作用）可防止 NG2 急性注射到脊髓引起的传导阻滞。鞘内输注 NG2-Ab，通过 然而，使用微型渗透泵 2 周后，运动功能仅得到有限且短暂的改善 继SCI。试图设计一种安全、长期且临床可行的 NG2- 递送方法 Ab，我们已经成功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 NG2 抗体：AAV-NG2Ab 的相关递送。 初步实验结果表明，AAV-NG2Ab 和 AAV-NT3 联合给药 与 ChABC/AAV-NT3 相比，在轻微（150 kDyn）挫伤后，诱导了更大的改善。效果 然而，这种新型基因疗法 (AAV-NG2Ab/AAV-NT3) 工具对大鼠运动恢复的作用仍然有限 轻度挫伤大鼠，重度挫伤 SCI 大鼠则不太明显。 试图进一步提高AAV-NG2Ab/AAV-NT3的有益效果，并将改进范围扩大到 对于严重的 SCI 模型，我们现在建议添加第三种治疗成分，即非侵入性重复电击 脊椎磁刺激 (rSEMS)。我们最近发现 rSEMS 增强了传输 并改善运动神经元突触输入处 NMDA 受体的功能，这是启动作用所必需的 NT-3 在这些输入端。因此，在拟议的项目中，我们设计了一种新的添加剂处理方法，包括 AAV10-NG2Ab、AAV10-NT3 和 rSEMS。除了轻度挫伤模型外，我们建议使用 严重的胸中部挫伤，已知会导致运动功能和膀胱活动的严重缺陷 在大鼠和人类 SCI 中。这项研究的一个重要而新颖的方面是对所提出的新基因的评估 治疗（AAV-NG2Ab/AAV-NT3）联合 rSEMS 对膀胱功能的影响。为了评估这些措施的功效 治疗时，我们会进行多学科检查，包括体内生理学、解剖学、 免疫化学和行为。我们将检查新疗法的效果（相加或协同） 治疗 (1) 加强通过损伤中心到腰椎运动神经元的突触连接，以及 然后是后肢肌肉（使用体内电生理学）； (2) 纤维的解剖可塑性占 接受这种新颖的治疗后突触反应的持续性（使用解剖学追踪和 共焦显微镜）； (3) 运动性能的恢复（使用自动猫步步态分析）； (4) 恢复膀胱活动（使用代谢室和膀胱测量/电生理学）。为了更好地理解 为了了解细胞水平治疗的效果，我们将研究轴突髓鞘再生（使用电子显微镜）和 NG2- 联系朗飞节点的正过程。正在进行的实验的初步结果显示了以下方面的改进 严重中胸挫伤性 SCI 后接受这种新型附加治疗的大鼠的运动功能。