School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
J Neurotrauma. 2011 Dec;28(12):2475-83. doi: 10.1089/neu.2011.1928.
Recombinant adeno-associated viral (rAAV) vectors expressing neurotrophic genes reduce neuronal death and promote axonal regeneration in central nervous system (CNS) injury models. Currently, however, use of rAAV to treat clinical neurotrauma is problematic because there is a delay in the onset of transgene expression. Using the adult rat retina and optic nerve (ON), we have tested whether rAAV gene therapy administered at the time of injury combined with short-term pharmacotherapy has synergistic effects that enhance neuronal survival and regeneration. The ON was transected and a 1.5 cm segment of autologous peripheral nerve (PN) was grafted onto the cut end. At this time, bicistronic rAAV2 encoding ciliary neurotrophic factor (CNTF) and green fluorescent protein (rAAV2-CNTF-GFP) was injected into the injured eye. To provide interim support for axotomized retinal ganglion cells (RGCs) during vector integration and therapeutic transgene expression, rCNTF protein and a cyclic adenosine monophosphate (cAMP) analogue (CPT-cAMP) were injected intravitreally 3 and 10 days postoperatively. For comparison, another rAAV2-CNTF-GFP group received two intravitreal saline injections 3 and 10 days after the PN-ON surgery. A further PN graft group received only postoperative intravitreal injections of rCNTF plus CPT-cAMP. After 4 weeks, regenerating RGCs were retrogradely labelled by applying fluorogold to the distal end of each PN graft. Compared to saline-injected animals, both RGC survival and axonal regrowth were significantly higher in the rCNTF and CPT-cAMP injected rAAV2-CNTF-GFP group; approximately one third of the RGC population survived axotomy, and 27% of these regrew an axon. These values were also higher than those obtained in rats that received only rCNTF plus CPT-cAMP injections. Therefore, we show for the first time that rAAV-mediated gene delivery at the time of, or just after, neurotrauma is most successful when combined with temporary post-injury trophic support, and is potentially a viable treatment strategy for patients after acute CNS injury.
重组腺相关病毒（rAAV）载体表达神经营养基因可减少中枢神经系统（CNS）损伤模型中的神经元死亡并促进轴突再生。然而，目前使用 rAAV 治疗临床神经创伤存在问题，因为转基因表达的延迟。我们使用成年大鼠视网膜和视神经（ON）测试了在损伤时给予 rAAV 基因治疗并结合短期药物治疗是否具有协同作用，以增强神经元存活和再生。ON 被横断，并且自体周围神经（PN）的 1.5cm 段被移植到切割端。此时，将双顺反子 rAAV2 编码睫状神经营养因子（CNTF）和绿色荧光蛋白（rAAV2-CNTF-GFP）注入受伤的眼睛。为了在载体整合和治疗性转基因表达期间为轴突切断的视网膜神经节细胞（RGC）提供临时支持，在手术后 3 和 10 天，将 rCNTF 蛋白和环磷酸腺苷（cAMP）类似物（CPT-cAMP）注入玻璃体内。为了比较，另一个 rAAV2-CNTF-GFP 组在 PN-ON 手术后 3 和 10 天接受两次玻璃体内生理盐水注射。仅接受术后玻璃体内 rCNTF 加 CPT-cAMP 注射的另一个 PN 移植物组。4 周后，通过将荧光金应用于每个 PN 移植物的远端来逆行标记再生的 RGC。与生理盐水注射的动物相比，rCNTF 和 CPT-cAMP 注射的 rAAV2-CNTF-GFP 组的 RGC 存活和轴突再生均显著更高；大约三分之一的 RGC 群体存活轴突切断，其中 27％的这些群体再生了轴突。这些值也高于仅接受 rCNTF 加 CPT-cAMP 注射的大鼠获得的值。因此，我们首次表明，在神经创伤时或刚刚在神经创伤后进行 rAAV 介导的基因传递，与临时损伤后营养支持相结合最成功，并且对于急性中枢神经系统损伤后的患者可能是一种可行的治疗策略。
