Gene delivery to injured peripheral nerves

建议书摘要/摘要 外周神经和脑神经先天缺失或损伤会造成灾难性后果。这是 尤其是面神经，因为面部功能的丧失会导致眨眼影响视力、鼻瓣 塌陷影响呼吸，嘴唇松弛影响发音和口腔能力，面部受损 对情感和社会福祉产生深远负面影响的言论。就像周围神经 手术能够再生，可以用来恢复关键的感觉和运动功能。在病人中 面瘫患者可以通过在上嘴唇植入自体神经来传递轴突来恢复微笑。 从健侧的面神经供体分支到瘫痪侧。在随后的手术中，游离肌肉 移植到瘫痪侧，并通过跨面神经移植物进行神经化。这么长的神经移植 手术失败率为20%-30%，被认为是长度依赖性轴突生长停滞的次要因素。 新出现的证据表明，长距离神经再生受到进行性疾病的阻碍 轴突切断后神经元和雪旺细胞中促再生转录因子的下调 在长神经移植物的远端。迫切需要新的治疗策略来改善 远距离周围神经再生。 我们的长期目标是利用基因治疗技术延长雪旺细胞的促再生状态。 和神经元，以增强轴突穿透长神经移植物。虽然腺相关病毒(AAV) 载体已经被用于向周围神经传递基因，到目前为止，转导效率仍然很高 仍然是次优的，面神经还没有得到专门的研究。此外，交付到横切 尚未对周围神经进行研究。最近，使用包含AAV的杂交基因递送载体 载体和内源纳米颗粒被称为外周小体(exo-AAV)已经显示出令人印象深刻 改善了对内耳感觉毛细胞和中枢神经系统神经元的基因传递。 这项拟议的研究的目标是表征雪旺蛋白转导效率的差异。 新出现的AAV和外源AAV载体横断面神经的细胞和轴突。我们假设 改进的荧光报告基因转基因将被证明用于轴突切断的面神经和 与AAV载体不同，外源AAV对雪旺细胞有明显的抑制作用。一种双纯合子转基因(DTG) 外周高浓度表达青色荧光蛋白的荧光报告鼠 雪旺细胞中的轴突和金星蛋白将用于高通量无斑点评估 载体转导效率。结果将为AAV或外源AAV载体在临床上的潜在应用提供信息 将基因定向输送到受伤的面部神经。

Proposal Summary/Abstract Congenital absence or injury to peripheral and cranial nerves yields devastating consequences. This is especially true of the facial nerve as loss of facial function leads to impaired blink impacting sight, nasal valve collapse impacting respiration, lip flaccidity impacting articulation and oral competence, and impaired facial expression with profound negative impacts on emotional and social well-being. As peripheral nerves are capable of regeneration, surgery may be employed to restore critical sensory and motor function. In patients with facial palsy, smile may be reanimated by interposing a nerve autograft across the upper lip to route axons from a healthy-side facial nerve donor branch to the paralyzed side. In a subsequent surgery, free muscle is transplanted into the paralyzed side and neurotized by the cross-facial nerve graft. Such long nerve grafting procedures carry a 20-30% failure rate, believed to be secondary to length-dependent axonal growth arrest. Emerging evidence suggests nerve regeneration over long distances is hampered by progressive downregulation of pro-regenerative transcription factors within axotomized neurons and Schwann cells residing in the distal portions of long nerve grafts. There is a critical need for novel therapeutic strategies to improve peripheral nerve regeneration over long distances. Our long-term goal is to employ gene therapy techniques to prolong pro-regeneration states of Schwann cells and neurons to enhance axonal penetration across long nerve grafts. Though adeno-associated virus (AAV) vectors have been employed for gene delivery to peripheral nerves, transduction efficiency has heretofore remained suboptimal and the facial nerve has not been specifically studied. Further, delivery to transected peripheral nerves has not been investigated. Recently, use of hybrid gene delivery vectors comprising AAV vectors and endogenous nanoparticles termed exosomes (exo-AAV) has demonstrated impressive improvement in gene delivery to sensory hair cells of the inner ear and neurons of the central nervous system. The goal of the proposed research is to characterize differences in the efficiency of transduction of Schwann cells and axons of transected facial nerve by emerging AAV and exo-AAV vectors. We hypothesize that improved fluorescent reporter transgene transduction will be demonstrated for axotomized facial neurons and Schwann cells transfected with exo-AAV as opposed to AAV vectors. A double homozygous transgenic (dTg) fluorescent reporter mouse expressing cyan fluorescent protein (CFP) at high concentrations in peripheral axons and Venus protein in Schwann cells will be employed for high-throughput stain-free assessment of vector transduction efficiency. Results will inform the potential clinical use of AAV or exo-AAV vectors for targeted gene delivery to injured facial nerves.