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: 7935772
• 负责人: PETER R BRINK
• 金额: $ 41.28万
• 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/7935772
• 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递送代表了独特的和潜在的临床重要的递送系统。我们以前的研究表明，缝隙连接组成的连接蛋白43（Cx43）是可渗透的siRNA和渗透的siRNA可以随后降低特定基因的mRNA水平。本文提出的研究的目的是表征siRNA从hMSC和其他通信能力细胞转移和渗透到靶组织中。以前我们确定由Cx43组成的缝隙连接转移siRNA，而由Cx 32或Cx 26组成的缝隙连接则不会。因此，siRNA的通道渗透性取决于连接蛋白。在目标1中，我们将确定由Cx40、Cx 37和Cx43组成的间隙连接对吗啉代和siRNA的渗透性。选择Cx43、Cx40和Cx 37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HCN 2和GFP的siRNA的合成和降解速率。我们还将研究膜蛋白，起搏器通道HCN 2功能沉默的有效性和时间过程。我们将通过表征经由间隙连接介导的siRNA从hMSC或其他通信感受态细胞递送至表达HCN 2的靶细胞的HCN 2的功能性沉默，来检验经由间隙连接通道的siRNA细胞递送可使靶细胞中的HCN 2通道功能沉默的假设。我们使用RT-PCR跟踪HCN 2 mRNA浓度，以允许在siRNA存在下估计随时间推移的相对含量。我们还将确定标记的吗啉代/siRNA的浓度，以建立沉默基因（HCN 2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，我们将推导出用于siRNA沿着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（hMSC）的数量和位置。在目标5中，我们将评估siRNA在体内沉默GFP中的有效性。我们使用裸小鼠，将表达GFP的1000万个细胞的推注注射到真皮中或肌内，随后在不同时间注射负载有靶向GFP的siRNA的hMSC。我们将使用整个动物成像随时间跟踪GFP荧光图像。 公共卫生相关性：小干扰RNA（siRNA）靶向单一蛋白质，降低其表达。因此，它具有作为高选择性药物的巨大潜力。然而，用于其体内递送的系统不是最佳的。本申请研究免疫豁免的成体间充质干细胞（MSC）以及其他细胞类型向靶细胞或组织递送小干扰RNA（siRNA）的能力。这种基于细胞的递送的基础是差距连接通道。这些通道连接偶联细胞的细胞内区室，并允许小分子转移而不进入细胞外空间。我们已经确定，制造连接蛋白（间隙连接的构建块）的细胞可以转移siRNA。该应用程序询问细胞是否可以作为siRNA的递送系统。通过实验和数学建模的组合，我们试图确定siRNA的细胞递送在体外和体内穿透组织的能力。