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: 10531535
• 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/10531535
• 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; 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.

自愿步行和膀胱功能障碍的能力受损是退伍军人的一个急性问题 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/再生抑制剂和（3）缺乏神经营养蛋白支持。使用动物模型，我们最近有 证明与疤痕相关的抑制性软骨素硫酸盐蛋白聚糖（CSPG）的降解与 酶软骨素酶-ABC（CHABC），结合基于AAV的神经营养蛋白NT3的递送 轻度挫伤科学后的部分改进。使用CHABC的潜在缺点是 不是特异性的，即降解所有CSPG，包括那些是细胞外基质的重要组成部分的CSPG。 在寻找更具体的目标时，我们最近证明了一个CSPG分子NG2， 被称为大脑和脊髓损伤后轴突再生的主要障碍 传导，但其他测试的CSPG没有。单克隆NG2函数中和急性给药 抗体（NG2-AB；最初旨在防止NG2对轴突生长的抑制作用）防止 NG2急性注射到脊髓中引起的传导阻滞。鞘内输注NG2-AB，通过 然而，渗透微型泵2周仅诱导运动功能的有限和短暂的改进 跟随科学。试图设计一种方法，以实现安全，延长和临床可行的NG2-的方法 AB，我们成功地创建了一种新的基于AAV-10的基于AAV-10的基因治疗工具，用于延长和临床 - 重组单链可变片段（SCFV）抗NG2抗体的相关递送：AAV-NG2AB。 初步实验的结果表明，AAV-NG2AB和AAV-NT3的组合施用 在轻度（150 kDyn）挫伤之后，与CHABC/AAV-NT3相比，诱导的改善更大。效果 然而，这种新型基因疗法（AAV-NG2AB/AAV-NT3）关于运动恢复的工具仍然有限 严重挫伤SCI的大鼠轻度挫伤，较明显。 试图进一步改善AAV-NG2AB/AAV-NT3的有益影响并扩大改进 严重的SCI模型，我们现在建议添加第三个治疗成分，即非侵入性重复的电 - 脊柱椎骨（RSEMS）的磁刺激。我们最近发现RSEM可以加强传播 并改善NMDA受体在运动神经元突触输入中的功能，这是启动的效果所必需的 在这些输入处的NT-3。因此，在拟议的项目中，我们设计了一种新的添加剂处理，包括 AAV10-NG2AB，AAV10-NT3和RSEMS。除了轻度的损伤模型外，我们建议使用 严重的中腹膜挫伤已知会引起运动功能和膀胱活性的主要缺陷 在老鼠和人类科学中。这项研究的一个重要而新颖的方面是评估提出的新基因 治疗（AAV-NG2AB/AAV-NT3）结合了膀胱功能的RSEM。评估这些功效 治疗方法，我们将进行多学科检查，包括体内生理学，解剖学， 免疫化学和行为。我们将研究新治疗的影响（添加剂或协同作用） （1）通过损伤震中加强突触连接到腰部运动神经元，以及 然后是后肢肌肉（使用体内电生理学）； （2）纤维的解剖塑性 暴露于这种新型治疗后突触反应的持久性（使用解剖学跟踪和 共聚焦显微镜）； （3）恢复运动性能（使用自动走秀步态分析）； （4） 膀胱活性的恢复（使用代谢室和囊肿/电生理学）。更好地理解 在细胞水平上的治疗作用，我们将研究轴突再生（使用电子显微镜）和NG2-- 接触兰维尔节点的积极过程。正在进行的实验的初步结果显示了 严重的胸膜中期后的SCI后，已经接受了这种新型添加剂治疗的大鼠的运动功能。