Tissue engineering with a complete RASSL toolbox

具有完整 RASSL 工具箱的组织工程

• 批准号: 7254898
• 负责人: Bruce R Conklin
• 金额: $ 61.47万
• 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 2008-09-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7254898
• 关键词: Abbreviations; Acute; Adverse effects; Agonist; Animals; Arrhythmia; Back; Binding; Blood Vessels; Bone Marrow; Brain; CD31 Antigens; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Transplants; Cells; Chimeric Proteins; Clinical; Complex; Development; Down-Regulation; Drug or chemical Tissue Distribution; ES Cell Line; Electrocardiogram; Embryo; Engineering; Engraftment; Fluorescence Microscopy; Future; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; GRK; GTP-Binding Proteins; Galactosidase; Gene Targeting; Genes; Genetic Engineering; Genomics; Grant; Green Fluorescent Proteins; Growth; Growth and Development function; Heart; Heart Rate; Heart failure; Hormonal; Hormones; Human; Hypoxia; Hypoxia Inducible Factor; Implant; In Vitro; Ischemia; Life; Ligand Binding Domain; Ligands; Location; Long-Term Effects; Melanocortin 4 Receptor; Methods; Movement Disorders; Mus; Muscle Cells; Mutate; Mutation; Nature; Neomycin; Opioid; Pathway interactions; Patients; Pattern; Peptide Receptor; Peptides; Personal Satisfaction; Pharmaceutical Preparations; Phosphotransferases; Physiological; Physiology; Positioning Attribute; Principal Investigator; Process; Recycling; Research; Resistance; Score; Series; Signal Transduction; Site; Solutions; Stem cells; Surface; System; Techniques; Testing; Therapeutic; Time; Tissue Engineering; Tissues; Transgenic Mice; Transgenic Organisms; Transplantation; Transplanted tissue; Tropomyosin; Variant; Vascular Endothelium; Ventricular Remodeling; base; cardiogenesis; cell type; concept; design; embryonic stem cell; extracellular; implantation; in vivo; kappa opioid receptors; motilin receptor; peptide I; peptide hormone; programs; promoter; prototype; receptor; relating to nervous system; response; small molecule; stem; success; tool; trafficking; ubiquitin ligase; vector

DESCRIPTION (provided by applicant): We propose to reengineer hormonal signaling systems to gain pharmacological control of the growth and development of cardiomyocytes and other potentially therapeutic cells. Although G protein coupled receptors (GPCRs) control a wide variety of physiologic responses, biologists currently lack such sophisticated tools to harness these same processes in vivo. As the field of tissue engineering matures, we need pharmacologically activated "on" and "off" switches to control the therapeutic tissues long after they have been transplanted back into the patient. We have used GPCRs to develop Receptors Activated Solely by Synthetic Ligands (RASSLs). These engineered receptors no longer respond to endogenous peptide hormones, but can still be activated by small-molecule drugs. Our prototype RASSL activates Gi and has been used to regulate heart rate and trigger ventricular remodeling in transgenic mice. Specific Aim 1. To control GPCR signaling in vivo, we will develop a series of RASSLs that activate each of the major G protein pathways (Gs, Gi, Gq). Each RASSL will also be fused to the green fluorescent protein (GFP), and will be altered at key regulatory sites, resulting in additional RASSLs that are either resistant or hypersensitive to downregulation. Specific Aim 2.To test the hypothesis that RASSL signaling can modulate physiological responses in transgenic mice, each RASSL will be targeted to the same cardiac-specific gene locus (Tropomyosin 1a) so as to be expressed at identical levels and locations in the mouse heart. RASSL-induced effects will be determined with short-term (ECG), and long-term (cardiac remodeling, cardiomyopathy) responses. Specific Aim 3. To test the effects of RASSL activation on growth and development, embryonic stem (ES) cell-derived cardiac myocytes will be examined that have RASSLs targeted to the four genomic loci: Tropomyosin 1a (cardiomyocyte-specific), PECAM (vascular endothelium specific), Hypoxia Inducible Factor 1a (ischemia induced) and Ubiquitin Ligase E2B (ubiquitously expressed). These aims provide a RASSL toolbox for tissue engineering. In the future, it is possible that RASSLs could be used in transplanted cells providing pharmacological control to enhance engraftment or reduce arrhythmias after implantation. These aims also will allow us to provide RASSLS to research colleagues who wish to use RASSLs in other tissues. Rapid gene targeting is possible since we will design RASSL targeting vectors to use "one-way" Lox sites that have been engineered into over 1200 genes in mouse ES cells. A complete set of RASSL targeting vectors will provide biologists with a RASSL toolbox to selectively activate any major GPCR pathway in a wide variety of tissues.

描述（由申请人提供）： 我们建议重新设计激素信号系统，以获得对心肌细胞和其他潜在治疗细胞的生长和发育的药理学控制。尽管 G 蛋白偶联受体 (GPCR) 控制着多种生理反应，但生物学家目前缺乏如此复杂的工具来在体内利用这些相同的过程。随着组织工程领域的成熟，我们需要药物激活的“开”和“关”开关来控制治疗组织在移植回患者体内很久之后。我们使用 GPCR 开发了仅由合成配体激活的受体 (RASSSL)。这些工程受体不再对内源性​​肽激素产生反应，但仍然可以被小分子药物激活。我们的原型 RASSL 可激活 Gi，并已用于调节转基因小鼠的心率和触发心室重塑。 具体目标 1. 为了控制体内 GPCR 信号传导，我们将开发一系列激活每个主要 G 蛋白途径（Gs、Gi、Gq）的 RASSL。每个 RASSL 还将与绿色荧光蛋白 (GFP) 融合，并在关键调控位点发生改变，从而产生对下调具有抗性或超敏感的额外 RASSL。 具体目标 2.为了测试 RASSSL 信号传导可以调节转基因小鼠生理反应的假设，每个 RASSL 将靶向相同的心脏特异性基因位点（原肌球蛋白 1a），以便在小鼠心脏中以相同的水平和位置表达。 RASSL 诱导的效应将通过短期（心电图）和长期（心脏重塑、心肌病）反应来确定。 具体目标 3. 为了测试 RASSL 激活对生长和发育的影响，将检查胚胎干 (ES) 细胞来源的心肌细胞，这些细胞的 RASSL 靶向四个基因组位点：原肌球蛋白 1a（心肌细胞特异性）、PECAM（血管内皮特异性）、缺氧诱导因子 1a（缺血诱导）和泛素连接酶 E2B（普遍表达）。 这些目标为组织工程提供了 RASSL 工具箱。将来，RASLS 有可能用于移植细胞，提供药理学控制，以增强植入或减少植入后的心律失常。这些目标还将使我们能够向希望在其他组织中使用 RASSL 的研究同事提供 RASSLS。快速基因靶向是可能的，因为我们将设计 RASSL 靶向载体以使用“单向”Lox 位点，这些位点已被工程化到小鼠 ES 细胞中的 1200 多个基因中。一套完整的 RASSL 靶向载体将为生物学家提供 RASSL 工具箱，以选择性激活各种组织中的任何主要 GPCR 通路。