Tissue Engineering with a Modular RASSL Toolbox

使用模块化 RASSL 工具箱进行组织工程

• 批准号: 7691304
• 负责人: Bruce R Conklin
• 金额: $ 64.6万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7691304
• 关键词: Abbreviations; Acute; Adopted; Adult; Adverse effects; Affect; Affinity; Arrhythmia; Artificial cardiac pacemaker; Biological; Biological Models; Bone Growth; Brain; Calcium; Cardiac; Cell Culture Techniques; Communities; Contracts; Cyclic AMP; Data; Disease; Echocardiography; Effectiveness; Electrocardiogram; Embryo; Engineering; Enhancers; Ensure; Figs - dietary; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; Genetic; Genome; Goals; Grant; Green Fluorescent Proteins; Growth and Development function; Health; Heart; Heart Rate; Hormones; Human; In Vitro; Individual; Knock-in Mouse; Lentivirus Vector; Ligands; Measurable; Measurement; Measures; Methods; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Muscarinic Acetylcholine Receptor; Muscle Cells; Mutagenesis; Opioid; Opioid Receptor; Pacemakers; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Phosphatidylinositols; Phospholipase C; Physiological; Physiological Processes; Production; Proteins; RNA; Research; Research Personnel; Research Project Grants; Series; Serotonin Receptors 5-HT4; Signal Pathway; Signal Transduction; Site; System; Tamoxifen; Taste Perception; Technology; Testing; Tetanus Helper Peptide; Tetracyclines; Time; Tissue Engineering; Tissues; Trans-Activators; Transgenic Mice; Transgenic Organisms; Work; base; bone; brain cell; embryo monitoring; embryonic stem cell; flexibility; functional restoration; heart pacemaker tissue; improved; in vivo; insight; interest; meetings; mortality; neurotransmission; next generation; promoter; prototype; public health relevance; receptor; receptor function; recombinase; release of sequestered calcium ion into cytoplasm; response; small molecule; stem; tool; vector; Abbreviations; Acute; Adopted; Adult; Adverse effects; Affect; Affinity; Arrhythmia; Artificial cardiac pacemaker; Biological; Biological Models; Bone Growth; Brain; Calcium; Cardiac; Cell Culture Techniques; Communities; Contracts; Cyclic AMP; Data; Disease; Echocardiography; Effectiveness; Electrocardiogram; Embryo; Engineering; Enhancers; Ensure; Figs - dietary; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; Genetic; Genome; Goals; Grant; Green Fluorescent Proteins; Growth and Development function; Health; Heart; Heart Rate; Hormones; Human; In Vitro; Individual; Knock-in Mouse; Lentivirus Vector; Ligands; Measurable; Measurement; Measures; Methods; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Muscarinic Acetylcholine Receptor; Muscle Cells; Mutagenesis; Opioid; Opioid Receptor; Pacemakers; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Phosphatidylinositols; Phospholipase C; Physiological; Physiological Processes; Production; Proteins; RNA; Research; Research Personnel; Research Project Grants; Series; Serotonin Receptors 5-HT4; Signal Pathway; Signal Transduction; Site; System; Tamoxifen; Taste Perception; Technology; Testing; Tetanus Helper Peptide; Tetracyclines; Time; Tissue Engineering; Tissues; Trans-Activators; Transgenic Mice; Transgenic Organisms; Work; base; bone; brain cell; embryo monitoring; embryonic stem cell; flexibility; functional restoration; heart pacemaker tissue; improved; in vivo; insight; interest; meetings; mortality; neurotransmission; next generation; promoter; prototype; public health relevance; receptor; receptor function; recombinase; release of sequestered calcium ion into cytoplasm; response; small molecule; stem; tool; vector

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.

描述（由申请人提供）： 我们建议创建一个柔性多重系统，用于表达多种工程受体，以促进组织的工程，例如心脏细胞，脑和骨骼。我们开发了新的G蛋白偶联受体（GPCR），称为RASSL（仅由合成配体激活）。 RASSL被设计为对内源激素无反应，但仍被具有纳摩尔亲和力的小分子药物激活，体内副作用相对较少，因此非常适合组织工程研究。我们的原型RASSL（RO1）激活GI信号通路，抑制cAMP的形成。当在特定组织中表达RO1时，它会影响多种生理过程，例如心率，心脏重塑，神经递质和骨骼生长。我们帮助建立了一个越来越多的研究人员社区，他们设计了新的RASSL，以激活所有主要的GPCR途径，包括GS，这增加了CACK组和GQ，并刺激了钙动员。现在，我们建议应对RASSL用户的下一个主要挑战。缺乏针对个体或多个RASSL的体内递送系统的强大且可控的，这阻碍了科学界更广泛地使用RASSL。为了满足这一需求，我们将使用一组可以模拟与这些特定目标的任何GPCR信号组合的共同组件开发一个灵活的系统来表达多个RASSL。 AIM 1。创建一个体内表达系统，以最佳的空间和时间控制RASSL表达。我们的最终目标是将CRE和TET系统结合起来以进行RASSL表达。我们将通过一系列三个向量独立评估每个策略。我们将在心脏起搏器中使用GI-RASSL作为具有强大体内反应的模型系统。目的2。表征对小鼠心脏起搏器组织和ES细胞中每个主要RASSL诱导的GPCR信号途径的生理反应。具有较高和低水平的基础信号传导的GS，GI-和GQ-RASSL将在心脏起搏器组织中表达。为了定义RASSL诱导的表型，我们使用了ES细胞衍生的肌细胞的细胞培养模型，以及在小鼠胚胎中开发起搏器组织和成年小鼠的心脏监测。目标3。确定共同刺激多个GPCR信号通路的生理效应。我们将使用2A多水系统来共表达RASSL的组合，以激活三个主要信号通路中的每一个。本系列中的四个信号组合将包括GS-GI，GS-GQ，GI-GQ和GS-GI-GQ。我们还将使用可以通过单个配体激活的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基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，例如心脏，脑和骨骼。 RASSL还为GPCR的作用提供了基本的见解，GPCR是许多广泛使用的药物的靶标。最后，我们将心脏起搏器用作模型系统。我们希望获得对心律不齐的基本见解，这是发病率和死亡率的主要原因。