SiRNA therapeutics: Gap junction delivery in vitro and in vivo

SiRNA 疗法：体外和体内间隙连接传递

• 批准号: 8136713
• 负责人: PETER R BRINK
• 金额: $ 41.07万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136713
• 关键词: 3-Dimensional; Address; Adult; Allogenic; Animals; Autologous; Bolus Infusion; Canis familiaris; Cell Count; Cell Line; Cells; Characteristics; Communication; Complementary DNA; Connexin 43; Connexins; Coupled; Data; Dermis; Diffuse; Diffusion; Dimensions; Effectiveness; Exposure to; Extracellular Space; Fluorescence; Gap Junctions; Gene Silencing; Gene Targeting; Genes; Giant Cells; Green Fluorescent Proteins; Heart; High Pressure Liquid Chromatography; Hour; Human; Image; Immune response; In Vitro; Injection of therapeutic agent; Knowledge; Length; Measures; Mediating; Membrane Proteins; Mesenchymal Stem Cells; Messenger RNA; Modeling; Monitor; Nude Mice; Organ; Pacemakers; Pathway interactions; Penetration; Permeability; Pharmaceutical Preparations; Positioning Attribute; Problem Solving; Proteins; Protocols documentation; RNA chemical synthesis; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Site; Small Interfering RNA; Source; System; Tail; Testing; Therapeutic; Therapeutic Agents; Time; Tissues; Veins; Western Blotting; base; cell motility; cell type; clinical application; density; design; fluorescence imaging; gap junction channel; heart cell; in vivo; inhibitor/antagonist; lucifer yellow; mathematical model; monolayer; patch clamp; public health relevance; research study; small molecule; three-dimensional modeling; time use; tumor

DESCRIPTION (provided by applicant): The cellular delivery of siRNA via gap junctions represents a unique and potentially clinically important delivery system. Our previous studies have shown that gap junctions composed of connexin43 (Cx43) are permeable to siRNAs and permeating siRNAs can subsequently reduce the mRNA levels of a specific gene. The aim of the studies proposed here is to characterize the transfer and permeation of siRNA from hMSCs and other communication competent cells into a target tissue. Previously we determined that gap junctions composed of Cx43 transfer siRNA, whereas those composed of Cx32 or Cx26 will not. Hence channel permeability for siRNA depends on the connexin. In aim 1 we will determine the permeability of gap junctions made of Cx40, Cx37 and Cx43, to morpholinos and siRNAs. Cx43, Cx40 and Cx37 are chosen because they are ubiquitously expressed in vivo in many organs. In aim 2 we will determine the synthesis and degradation rates of siRNA targeting HCN2 and GFP. We will also investigate the efficacy and time course of functional silencing of the membrane protein, the pacemaker channel HCN2. We will test the hypothesis that cellular delivery of siRNA via gap junction channels can silence HCN2 channel function in target cells by characterizing the functional silencing of HCN2 via gap junction mediated delivery of siRNA from hMSCs or other communication competent cells to a target cell expressing HCN2. We follow HCN2 mRNA concentration using RT-PCR to allow an estimate of the relative content over time in the presence of siRNA. We will also determine the concentrations of tagged morpholinos/siRNAs to establish the effective concentration necessary to silence a gene (HCN2 or GFP) and also provide parameters for our 2D/3D model to determine penetration within a tissue. In aim 3 we will experimentally assess how far siRNA can penetrate multiple cell layers of a syncytium. In aim 4 we will derive a model for transfer of siRNA along a simple linear chain of cells or geometries in 2 or 3 dimensions. It will be used to predict the number and position of siRNA containing cells (hMSCs) required to silence function in a tissue or an organ/tumor. In aim 5 we will assess siRNA effectiveness in silencing GFP in vivo. We use nude mice and inject a bolus of 10 million cells expressing GFP into the dermis or intramuscularly followed at various times with an injection of hMSCs loaded with siRNA targeting GFP. We will track the GFP fluorescence image over time using whole animal imaging. PUBLIC HEALTH RELEVANCE: Small interfering RNA (siRNA) targets a single protein reducing its expression. As such it has great potential as a highly selective drug. However systems for its in vivo delivery are not optimal. The present application investigates the ability of the immuno-privileged adult mesenchymal stem cell (MSC) as well as other cell types to deliver small interfering RNA (siRNA) to a target cell or tissue. The basis of this cell based delivery is the gap junction channel. These channels connect the intracellular compartments of coupled cells and allow transfer of small molecules without entry into the extracellular space. We have already established that cells that make connexins (the building block of gap junctions) can transfer siRNAs. This application asks whether cells can serve as a delivery system for siRNA. By a combination of experiment and mathematical modeling we seek to determine the ability of cellular delivery of siRNA to penetrate tissues in vitro and in vivo.

描述（由申请人提供）：siRNA通过间隙连接的细胞传递是一种独特的、具有潜在临床重要性的传递系统。我们之前的研究表明，由connexin43 （Cx43）组成的间隙连接可以渗透到sinas中，渗透的sinas随后可以降低特定基因的mRNA水平。本文提出的研究目的是表征siRNA从hMSCs和其他通信能力细胞转移和渗透到靶组织中的过程。先前我们确定由Cx43组成的间隙连接可以转移siRNA，而由Cx32或Cx26组成的间隙连接则不能。因此siRNA的通道通透性取决于连接蛋白。在目的1中，我们将确定由Cx40、Cx37和Cx43组成的间隙连接对morpholinos和sirna的通透性。之所以选择Cx43、Cx40和Cx37，是因为它们在体内许多器官中普遍表达。在目标2中，我们将确定靶向HCN2和GFP的siRNA的合成和降解率。我们还将研究功能性沉默膜蛋白（起搏器通道HCN2）的疗效和时间过程。我们将通过表征HCN2的功能性沉默，通过间隙连接介导的siRNA从hMSCs或其他通信能力强的细胞传递到表达HCN2的靶细胞，来验证通过间隙连接通道传递siRNA可以沉默靶细胞中HCN2通道功能的假设。我们使用RT-PCR跟踪HCN2 mRNA浓度，以便在siRNA存在的情况下估计相对含量随时间的变化。我们还将确定标记的morpholinos/ sirna的浓度，以建立沉默基因（HCN2或GFP）所需的有效浓度，并为我们的2D/3D模型提供参数，以确定组织内的渗透。在目标3中，我们将通过实验评估siRNA可以穿透合胞体的多个细胞层的程度。在目标4中，我们将推导出siRNA沿简单线性细胞链或二维或三维几何结构转移的模型。它将用于预测组织或器官/肿瘤中沉默功能所需的含siRNA细胞（hMSCs）的数量和位置。在目标5中，我们将评估siRNA在体内沉默GFP的有效性。我们使用裸鼠，将1000万个表达GFP的细胞注射到真皮或肌肉内，然后在不同时间注射装载靶向GFP的siRNA的hMSCs。我们将使用全动物成像跟踪GFP荧光图像随时间的变化。