Tissue Engineering with a Modular RASSL Toolbox

描述（由申请人提供）： 我们建议创建一个灵活的多顺反子系统，用于表达多种工程受体，以促进组织工程，如心脏细胞，脑和骨。我们开发了一类新的G蛋白偶联受体（GPCR），称为RASSL（仅由合成配体激活的受体）。RASSL被工程化为对内源性激素无反应，但仍被具有纳摩尔亲和力和相对较少的体内副作用的小分子药物激活，使其理想地适用于组织工程研究。我们的原型RASSL（RO 1）激活Gi信号通路，抑制cAMP的形成。当RO 1在特定组织中表达时，它可以影响多种生理过程，如心率，心脏重塑，神经传递和骨生长。我们已经帮助建立了一个不断增长的研究人员社区，他们设计了新的RASSL来激活所有主要的GPCR途径，包括增加cAMP形成的Gs和刺激钙动员的Gq。我们现在建议解决RASSL用户的下一个主要挑战。缺乏用于单个或多个RASSL的稳健且可控的体内递送系统阻碍了科学界对RASSL的更广泛使用。为了满足这一需求，我们将开发一种灵活的系统来表达多种RASSL，使用一组通用组件来模拟具有这些特定目标的任何GPCR信号组合。目标1.建立一个体内表达系统，以优化RASSL表达的空间和时间控制。我们的最终目标是将联合收割机Cre和泰特系统结合起来用于RASSL表达。我们将在一系列三个向量中独立评估每个策略。我们将在心脏起搏器中使用Gi-RASSL作为具有稳健体内反应的模型系统。目标2.研究小鼠心脏起搏组织和ES细胞对RASSL诱导的GPCR信号通路的生理反应。具有高和低水平基础信号传导的Gs-、Gi-和Gq-RASSL将在心脏起搏器组织中表达。为了确定RASSL诱导的表型，我们使用ES细胞来源的收缩肌细胞的细胞培养模型，以及在小鼠胚胎中发育起搏器组织和成年小鼠中的心脏监测。目标3.确定共刺激多个GPCR信号通路的生理效应。我们将使用2A多顺反子系统共表达RASSL的组合，激活三个主要信号通路中的每一个。本系列中的四种信令组合将包括Gs-Gi、Gs-Gq、Gi-Gq和Gs-Gi-Gq。我们还将使用可以被单个配体激活的RASSL，以确保共刺激。 公共卫生相关性声明（由申请人提供）：该项目对人类健康的根本重要性是为组织工程师提供强大的新工具，他们正在努力恢复许多组织的功能，如心脏，大脑和骨骼。RASSL还提供了对GPCR作用的基本见解，GPCR是许多广泛使用的药物的靶点。最后，我们使用心脏起搏器作为模型系统。我们希望获得对心律失常的基本见解，心律失常是发病率和死亡率的主要原因。

DESCRIPTION (provided by applicant): We propose to create a flexible polycistronic system for expressing multiple engineered receptors to facilitate the engineering of tissues, such as cardiac cells, brain, and bone. We developed new class of G-protein- coupled receptors (GPCRs) that are called RASSLs (Receptors Activated Solely by a Synthetic Ligand). RASSLs are engineered to be unresponsive to endogenous hormones, yet still activated by small-molecule drugs with nanomolar affinities and relatively few side effects in vivo, making them ideally suited for tissue- engineering studies. Our prototype RASSL (RO1) activates the Gi signaling pathway, inhibiting cAMP formation. When RO1 is expressed in specific tissues, it can affect diverse physiological processes, such as heart rate, remodeling of heart, neurotransmission, and bone growth. We have helped to establish a growing community of researchers who have engineered new RASSLs to activate all the major GPCR pathways, including Gs, which increases cAMP formation, and Gq, which stimulates calcium mobilization. We now propose to tackle the next major challenge for RASSL users. The lack of robust and controllable in vivo delivery systems for individual or multiple RASSLs has hindered wider use of RASSLs by the scientific community. To meet this need, we will develop a flexible system for expressing multiple RASSLs, using a set of common components that will mimic any GPCR signaling combination with these specific aims. Aim 1. To create an in vivo expression system for optimal spatial and temporal control of RASSL expression. Our ultimate goal is to combine Cre and Tet systems for RASSL expression. We will evaluate each strategy independently in a series of three vectors. We will use the Gi-RASSL in the cardiac pacemaker as a model system with robust in vivo responses. Aim 2. To characterize the physiological responses to each major RASSL-induced GPCR-signaling pathway in mouse cardiac pacemaker tissue and ES cells. Gs-, Gi-, and Gq-RASSLs with high and low levels of basal signaling will be expressed in cardiac pacemaker tissue. To define RASSL-induced phenotypes, we use a cell-culture model of ES cell-derived contracting myocytes, as well as developing pacemaker tissue in mouse embryos and cardiac monitoring in adult mice. Aim 3. To determine the physiological effects of co-stimulating multiple GPCR signaling pathways. We will use the 2A polycistronic system to co-express combinations of RASSLs that activate each of the three major signaling pathways. The four signaling combinations in this series will include Gs-Gi, Gs-Gq, Gi-Gq, and Gs-Gi-Gq. We will also use RASSLs that can be activated by a single ligand, so as to insure co-stimulation. Public Health Relevance Statement (Provided by Applicant): The fundamental importance of this project to human health is to provide powerful new tools for tissue engineers, who are working to restore function to many tissues, such as the heart, brain and bone. RASSLs also provide basic insights into the actions of GPCRs that are the targets of many widely used pharmaceuticals. Finally we are using the cardiac pacemaker as a model system. We hope to gain fundamental insights into cardiac arrhythmias that are a major cause of morbidity and mortality.