SiRNA therapeutics: Gap junction delivery in vitro and in vivo

描述(由申请人提供)：通过缝隙连接的细胞递送siRNA代表了一种独特且具有潜在临床重要性的递送系统。我们以前的研究表明，由连接蛋白43(Cx43)组成的缝隙连接对siRNAs具有渗透性，渗透siRNAs可以降低特定基因的mRNA水平。本研究的目的是研究siRNA从人骨髓间充质干细胞和其他具有通讯功能的细胞向靶组织的转移和渗透。以前我们确定由Cx43组成的缝隙连接传递siRNA，而由Cx32或Cx26组成的缝隙连接不传递siRNA。因此，siRNA的通道通透性依赖于连接蛋白。在目标1中，我们将测定由Cx40、Cx37和Cx43组成的缝隙连接对吗啉和siRNA的通透性。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，我们将测定针对HCN2和GFP的siRNA的合成和降解率。我们还将研究膜蛋白，即起搏器通道HCN2的功能沉默的有效性和时间过程。我们将通过缝隙连接介导的siRNA从hMSCs或其他有通讯能力的细胞向表达HCN2的靶细胞传递siRNA来表征HCN2的功能沉默，从而验证这样的假设：通过缝隙连接通道的细胞传递siRNA可以沉默靶细胞中的HCN2通道功能。我们使用RT-PCR跟踪HCN2mRNA的浓度，以便在siRNA存在的情况下估计随时间的相对含量。我们还将确定标记的吗啉/siRNAs的浓度，以确定沉默基因所需的有效浓度(HCN2或GFP)，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，我们将推导出一个模型，用于沿简单的线性细胞链或2或3维几何结构转移siRNA。它将被用来预测组织或器官/肿瘤中沉默功能所需的含有siRNA的细胞(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中，然后在不同的时间注射装载有针对GFP的siRNA的hMSCs。我们将使用整体动物成像跟踪GFP荧光图像随时间的变化。 与公共卫生相关：小干扰RNA(SiRNA)针对的是一种降低其表达的单一蛋白质。因此，它作为一种高度选择性的药物具有巨大的潜力。然而，其体内递送系统并不是最优的。本申请研究免疫特权的成人间充质干细胞(MSC)以及其他类型的细胞向靶细胞或组织递送小干扰RNA(SiRNA)的能力。这种基于细胞的传递的基础是缝隙连接通道。这些通道连接耦合细胞的细胞内隔间，允许小分子的转移而不进入细胞外空间。我们已经证实，制造连接蛋白(缝隙连接的构件)的细胞可以转移siRNA。这个应用程序询问细胞是否可以作为siRNA的传递系统。通过实验和数学模型的结合，我们试图确定siRNA在体外和体内穿透组织的细胞递送能力。

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.