Gene delivery to injured peripheral nerves

提案摘要/摘要 周围神经和颅神经的先天缺失或损伤会产生毁灭性的后果。这是 尤其是面部神经，因为面部功能的丧失导致眨眼受损，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 这对情绪和社会福祉产生了深刻的负面影响。由于周围神经 能够再生，手术可以用来恢复关键的感觉和运动功能。患者 对于面神经麻痹患者，可以通过在上唇插入自体神经移植物来引导轴突， 从健康侧的面神经供体分支到瘫痪侧。在随后的手术中， 移植到瘫痪侧，并通过跨面神经移植神经化。这么长的神经移植 手术具有20-30%的失败率，据信这是继发于长度依赖性轴突生长停滞。 新出现的证据表明，长距离的神经再生受到渐进性 下调轴突切断的神经元和雪旺细胞内的促再生转录因子， 在长神经移植物的远端部分。迫切需要新的治疗策略来改善 长距离的外周神经再生 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过长神经移植物。虽然腺相关病毒（AAV） 载体已被用于将基因递送至外周神经，迄今为止， 仍然是次优的，面部神经还没有被专门研究。此外，递送至横断的 尚未研究外周神经。最近，使用包含AAV的杂合基因递送载体， 载体和称为外泌体（exo-AAV）的内源性纳米颗粒已经证明了令人印象深刻的 基因传递到内耳感觉毛细胞和中枢神经系统神经元的改善。 这项研究的目的是描述雪旺氏细胞转导效率的差异。 细胞和轴突的横切面神经的出现AAV和exo-AAV载体。我们假设 将证明对轴突切断的面神经元的荧光报告基因转基因转导的改善， 与AAV载体相反，用exo-AAV转染的许旺细胞。双纯合转基因（dTg） 在外周血中高浓度表达青色荧光蛋白（CFP）的荧光报告小鼠 轴突和Venus蛋白将用于高通量无染色评估， 载体转导效率。结果将告知AAV或exo-AAV载体的潜在临床用途， 定向基因输送到受损的面部神经。

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.