Using silk as a biocompatible viral delivery system in the brain

 描述(申请人提供)：病毒表达载体被广泛用于促进或抑制特定基因的表达。在光遗传学研究中，病毒可以表达通道视紫红质-2或卤视紫质，从而实现对神经元活动的光学调节。病毒还可以表达基因编码的钙和电压指示器，以便对神经元活动进行光学监测。但目前的方法存在局限性。在这里，我们开发了基于丝素的腺相关病毒(AAV)的传递方法，以改善表达的定位，减少免疫原性反应，并提高转导效率。丝素是一种生物相容材料，当植入组织中时，可以溶解以释放病毒。我们已经发现，丝素/AAV可以用来表达光学电极末端的蛋白质。这导致了与表达区域的对准，并消除了注射AAV的第二次手术的需要。这比现有的方法更简单，有望增加吞吐量，导致更可靠的实验结果，并大大减少动物数量和所需的实验次数。我们将调整加工条件以改变丝膜的性质，以控制释放速度，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Silk/AAV是否可以减少炎症反应。注射AAVs可导致反应性胶质增生，这与扰乱突触特性有关。丝素可以保护病毒免受宿主免疫反应的影响，防止病毒降解，提高转导效率。我们将比较蚕丝/AAV混合物和常规注射AAV的反应性胶质化和突触特性。我们还将在活体成像(使用Silk/AAV涂层内窥镜)和光遗传学(使用Silk/AAV涂层光学电极)研究中评估Silk/AAV依赖表达的性能。另一个主要目标是在大范围内获得大部分细胞的有效转导。我们将确定是否可以将丝素/AAV薄片构图并放置在大脑表面，以在特定的皮质区域获得广泛表达。我们还建议使用丝素/AAV颗粒来获得广泛的表达。我们的初步实验表明，某些丝素/AAV制剂比单独注射病毒产生的表达更强、更广泛。我们将开发一种方法，通过在大脑中植入预制丝/AAV小颗粒来获得类似的表达模式。我们将评估Silk/AAV在救援实验中的效用，这些实验需要在一大部分细胞中广泛表达。我们还将确定Silk在大脑皮层神经元中表达GCaMP以进行成像的效用。如果丝绸可以用来获得 无论是局部或广泛的病毒表达，它都将是一项重大的技术进步，将对光遗传方法的应用以及更广泛的病毒传递做出重要贡献。

 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.