Using silk as a biocompatible viral delivery system in the brain

使用丝作为大脑中的生物相容性病毒传递系统

• 批准号: 8951722
• 负责人: WADE G REGEHR
• 金额: $ 25.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8951722
• 关键词: Animals; Area; Biocompatible; Biocompatible Materials; Brain; Calcium; Cell Fraction; Cells; Cerebellum; Corpus striatum structure; Dependovirus; Drug Formulations; Encapsulated; Endoscopes; Engineering; Fibroins; Film; Gene Expression; Genes; Gliosis; Goals; Halorhodopsins; Image; Immune response; Implant; Inflammatory Response; Injection of therapeutic agent; Lead; Methods; Monitor; Neurons; Operative Surgical Procedures; Optics; Pattern; Performance; Population; Process; Property; Proteins; Regulation; Silk; Solubility; Surface; Surgeon; Synapses; System; Techniques; Tissues; Viral; Viral Proteins; Virion; Virus; Water; Work; base; copolymer; expression vector; gene therapy; hydrogel film; immunogenic; improved; in vivo; in vivo imaging; interest; optogenetics; prevent; protein expression; public health relevance; research study; response; transduction efficiency; voltage

 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.

 描述（由申请人提供）：病毒表达载体广泛用于促进或敲低特定基因的表达。在光遗传学研究中，病毒可以表达视紫红质通道蛋白2或盐视紫红质，以允许神经元活动的光学调节。病毒还可以表达基因编码的。钙和电压指标允许光学监测神经元活动，但目前的方法有局限性，我们开发了基于丝的腺相关病毒（AAV）的传递方法，以改进。丝是一种生物相容性材料，当植入组织中时，丝/AAV 可用于在光极尖端表达蛋白质。这导致了与表达区域的对齐，并且无需进行第二次手术来注射 AAV，这比现有方法更简单，并且有望提高通量，带来更可靠的实验结果，并大大减少动物数量。以及所需的实验次数。我们将调整加工条件以改变丝膜的特性，以控制释放速率，以获得可靠的局部表达和不需要的表达。我们还将确定注射 AAV 是否​​会减少炎症反应，从而导致反应性神经胶质增生。 Silk 可能会扰乱突触特性，从而防止病毒降解并提高转导效率。我们将比较 Silk/AAV 混合物和传统注射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 涂层内窥镜）和光遗传学（使用丝/AAV 涂层光极）研究中的性能，另一个主要目标是获得 AAV 的有效转导。我们将确定是否可以将丝/AAV 薄片图案化并放置在大脑表面，以获得在特定皮质区域的广泛表达。获得我们的初步实验表明，某些 Silk/AAV 制剂比单独注射病毒产生的表达更强、更广泛。我们将开发通过在大脑中植入小型预制 Silk/AAV 颗粒来获得类似表达模式的方法。我们将评估 Silk/AAV 在需要在大部分细胞群中广泛表达的拯救实验中的效用，我们还将确定 Silk 在皮质神经元中表达 GCaMP 的效用，以进行成像。 无论是局部还是广泛的病毒表达，它都将代表一项重大的技术进步，将为光遗传学方法的应用以及更广泛的病毒传递做出重要贡献。