Using silk as a biocompatible viral delivery system in the brain

 描述（由申请人提供）：病毒表达载体广泛用于促进或敲减特定基因的表达。在光遗传学研究中，病毒可以表达通道视紫红质-2或盐视紫红质以允许神经元活性的光学调节。病毒还可以表达遗传编码的钙和电压指示剂，以允许光学监测神经元活动。但目前的方法有局限性。在这里，我们开发了腺相关病毒（AAV）的基于丝的递送方法，以改善表达的定位，减少免疫原性应答并提高转导效率。丝是一种生物相容性材料，当植入组织时可以溶解释放病毒。我们已经发现，丝/AAV可用于在光极尖端表达蛋白质。这导致与表达区域对齐，并且避免了注射AAV的第二次手术的需要。这比现有的方法更简单，并有望增加通量，导致更可靠的实验结果，并大大减少所需的动物数量和实验数量。我们将调整加工条件以改变丝膜的性质，从而控制释放速率，以获得可靠的局部表达并消除不需要的表达。我们还将确定丝/AAV是否减少炎症反应。注射AAV可导致反应性神经胶质增生，这与扰乱突触特性有关。丝可以保护病毒免受宿主免疫反应的影响，防止降解并提高转导效率。我们将比较丝/AAV混合物和常规注射AAV的反应性神经胶质增生和突触特性。我们还将评估丝/AAV依赖性表达在体内成像（使用丝/AAV涂覆的内窥镜）和光遗传学（使用丝/AAV涂覆的光极）研究中的性能。另一个主要目标是在大区域上获得大部分细胞的有效转导。我们将确定丝/AAV薄片是否可以被B图案化并放置在脑表面上以在限定的皮质区域中获得广泛表达。我们还建议使用丝/AAV颗粒来获得广泛表达。我们的初步实验表明，丝/AAV的一些制剂产生比单独注射病毒产生的更强和更广泛的表达。我们将开发通过在脑中植入小的预制丝/AAV颗粒来获得类似表达模式的方法。我们将评估丝/AAV在需要在大部分细胞群体中广泛表达的拯救实验中的效用。我们还将确定丝在皮质神经元中表达GCaMP用于成像的效用。如果丝可以用来获得 无论是局部的还是广泛的病毒表达，它都将代表一个主要的技术进步，这将对光遗传学方法的应用以及更一般地对病毒递送做出重要贡献。

 DESCRIPTION (provided by applicant): Viral expression vectors are widely used to either promote or knockdown the expression of specific genes. In optogenetic studies, viruses can express channelrhodopsin-2 or halorhodopsin to allow optical regulation of neuronal activity. Viruses can also express genetically encoded calcium and voltage indicators to allow optical monitoring of neuronal activity. But current methods have limitations. Here we develop methods for silk-based delivery of adeno-associated virus (AAV) in order to improve the localization of expression, to reduce immunogenic responses and to improve transduction efficiency. Silk is a biocompatible material that when implanted into tissue can dissolve to release viruses. We have found that silk/AAV can be used to express proteins at the tip of an optrode. This leads to alignment with the area of expression and obviates the need of a second surgery to inject AAV. This is simpler than existing methods and promises to increase throughput, lead to more reliable experimental results, and greatly reduce the number of animals and number of experiments required. We will adjust processing conditions to vary the properties of silk films to control the rates of release in order to obtain reliable localized expression and eliminate unwanted expression. We will also determine if silk/AAV reduces inflammatory responses. Injecting AAVs can lead to reactive gliosis, which has been implicated in perturbing synaptic properties. Silk may shield viruses from host immune responses, preventing degradation and improving transduction efficiency. We will compare reactive gliosis and synaptic properties for silk/AAV mixtures and conventional injections of AAV. We will also evaluate the performance of silk/AAV-dependent expression in in vivo imaging (using silk/AAV-coated endoscopes) and optogenetic (using silk/AAV-coated optrodes) studies. Another major goal is to obtain efficient transduction of a large fraction of cells over large regions. We will determine if thin sheets of silk/AAV can b patterned and placed on the surface of the brain to obtaining widespread expression in a defined cortical region. We also propose to use silk/AAV particles to obtain widespread expression. Our preliminary experiments suggest that some formulations of silk/AAV produce stronger and more widespread expression than that produced by injection of virus alone. We will develop approaches to obtain similar expression patterns by implanting small prefabricated silk/AAV particles in the brain. We will assess the utility of silk/AAV in rescue experiments that require widespread expression in a large fraction of a population of cells. We will also determine the utility of silk to express GCaMP in cortical neurons for imaging. If silk can be used to obtain either localized or widespread viral expression it will represent a major technical advance that will make an important contribution to the application of optogenetic approaches and more generally to viral delivery.