Parallel gene delivery from spinal cord bridges

描述（由申请人提供）：脊髓损伤导致损伤水平以下的瘫痪，这是由神经元和少突胶质细胞死亡、轴突缺失、脱髓鞘以及严重的脊髓神经元再生能力有限引起的。虽然脊髓神经元具有天生的再生能力，但它们受到促进再生的因子供应不足和抑制再生的因子供应充足的限制。我们的长期目标是开发一种基于生物材料的联合疗法，该生物材料桥接损伤部位以控制微环境，这是通过桥接微观结构和局部基因递送来提供刺激和指导再生的因素来实现的。该提议开发了用于脊髓再生的多通道桥，其能够从每个通道空间控制地递送一种或多种神经营养因子编码质粒。DNA将被固定在桥的通道内，以阻止辅助细胞浸润桥，并诱导神经营养因子的表达，所述神经营养因子将启动轴突伸长进入和穿过通道。另外，每个通道可以装载不同的神经营养蛋白编码质粒或质粒的组合，以针对特定的神经束定制通道。该假设基于以下观察：i）DNA递送可在体内诱导持续的、局部的转基因表达，ii）神经营养因子递送至损伤部位可促进轴突伸长成合成桥，iii）细胞移植或渗透泵植入不提供用于促进再生的神经营养因子的可控浓度，和iv）脊髓包含位于特定区域的多个神经束，并且包含不同的神经元类型。基于这些观察，实验的重点是设计用于有效基因转移的桥。这些研究被细分为3个特定目的：1）研究底物介导的DNA递送至桥通道并表征转基因表达（量、持续时间）和细胞转染（数量）。2)检验质粒诱导通道内神经营养因子分泌促进体外通道内特定神经元群体的局部存活的假设。3)检验以下假设：体内通道内神经营养因子的差异表达将促进轴突延伸到特定神经束的通道中。空间调控基因传递的发展对于再生复杂的组织结构（例如在脊髓内观察到的）将是重要的。这些结果将广泛影响组织工程，因为复杂组织结构的再生是一个基本问题。

DESCRIPTION (provided by applicant): Injury to the spinal cord results in paralysis below the level of the injury, which results from neuron and oligodendrocyte cell death, axonal loss, demyelination, and critically, the limited capacity of the spinal cord neurons to regenerate. Although spinal cord neurons have the innate capacity to regenerate, they are limited by an insufficient supply of factors to promote regeneration, and an abundant supply of factors that inhibit regeneration. Our long-term goal is to develop a combination therapy based on biomaterials that bridge the injury site to control the microenvironment, which is achieved through the bridge microstructure and localized gene delivery to provide factors that stimulate and direct regeneration. This proposal develops multiple channel bridges for spinal cord regeneration that are capable of spatially controlled delivery of one or more neurotrophin encoding plasmids from each channel. DNA will be immobilized within the channels of the bridge to transfect accessory cells infiltrating the bridge, and induce the expression of neurotrophic factors that will initiate axonal elongation into and across the channel. Additionally, each channel can be loaded with a different neurotrophin encoding plasmid, or combination of plasmids, to tailor the channel for specific neural tracts. This hypothesis is based on the observations that i) DNA delivery can induce sustained, localized transgene expression in vivo, ii) neurotrophin delivery to the injury site can promote axonal elongation into a synthetic bridge, iii) cell transplantation or osmotic pump implantation does not provide a controllable concentration of neurotrophins for promoting regeneration, and iv) the spinal cord contains multiple tracts that are located at specific regions and contain different neuronal types Based on these observations, the experimental focus is on designing bridges for efficient gene transfer. These studies are subdivided into 3 specific Aims: 1) Investigate substrate-mediated DNA delivery to the channels of the bridge and characterize transgene expression (quantity, duration) and cell transfection (number). 2) Test the hypotheses that plasmids inducing neurotrophin secretion within the channels promotes localized survival of specific neuronal populations within the channel in vitro. 3) Test the hypothesis that differential expression of neurotrophins within the channels in vivo will promote axonal extension into the channels for specific neural tracts. The development of spatially regulated gene delivery will be important for regenerating complex tissue architectures, such as that observed within the spinal cord. These results will broadly impact tissue engineering, as the regeneration of complex tissue architectures is a fundamental issue.