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: 9062539
• 负责人: WADE G REGEHR
• 金额: $ 21.19万
• 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/9062539
• 关键词: Animals; Area; Biocompatible; Biocompatible Materials; Brain; Calcium; Cell Fraction; Cells; Cerebellum; Corpus striatum structure; Dependovirus; Encapsulated; Endoscopes; Engineering; Fibroins; Film; Formulation; Gene Expression; Genes; Gliosis; Goals; Halorhodopsins; Health; Hydrogels; 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; immunogenic; improved; in vivo; in vivo imaging; interest; knock-down; optogenetics; prevent; protein expression; 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（由适用提供）：病毒表达载体被广泛用于促进或敲除特定基因的表达。在光遗传学研究中，病毒可以表达通道Ropopsin-2或卤代汀，以允许对神经元活性进行光学调节。病毒还可以表达一般编码的钙和电压指标，以允许对神经元活性进行光学监测。但是当前的方法有局限性。在这里，我们开发了基于丝绸相关病毒（AAV）的基于丝绸递送的方法，以改善表达的定位，减少免疫原性反应并提高传播效率。丝是一种生物相容性的材料，植入组织时可以溶解以释放病毒。我们发现，丝绸/AAV可用于在Optrode的尖端表达蛋白质。这会导致与表达区域的排列，并消除了第二次手术注射AAV的需要。这比现有方法更简单，并承诺增加吞吐量，导致更可靠的实验结果，并大大减少动物的数量和所需的实验数量。我们将调整加工条件，以改变丝绸膜的特性，以控制释放速率，以获得可靠的局部表达并消除不需要的表达。我们还将确定丝绸/AAV是否会减少炎症反应。注射AAV可以导致反应性神经胶质病，这在扰动突触特性中暗示。丝绸可能会屏蔽宿主免疫调查，以防止降解并提高转导效率。我们将比较丝绸/AAV混合物和常规注射AAV的反应性神经胶质性和突触特性。我们还将在体内成像（使用丝绸/AAV涂层内窥镜）和光遗传学（使用丝绸/AAV涂层的Optrododes研究）中评估丝/AAV依赖性表达的性能。另一个主要目标是在大型区域中有效地转移大部分细胞。我们将确定丝绸/AAV的薄片是否可以B形成图案并放在大脑的表面上，以获得定义的皮质区域的宽度表达。我们还建议使用丝绸/AAV颗粒获得宽度表达。我们的初步实验表明，与单独注射病毒相比，丝绸/AAV的某些公式产生的表达更强，更广泛。我们将通过在大脑中实现小的预制丝/AAV颗粒来开发方法来获得相似的表达模式。我们将评估丝绸/AAV在救援实验中的效用，这些实验需要大量细胞群中的宽度表达。我们还将确定丝绸在皮质神经元中表达GCAMP的实用性进行成像。如果可以使用丝绸获取 局部或广泛的病毒表达将代表一个重大的技术进步，这将为光遗传学方法的应用做出重要贡献，并且更普遍地对病毒递送。