Tissue engineering with a complete RASSL toolbox

具有完整 RASSL 工具箱的组织工程

• 批准号: 6898907
• 负责人: Bruce R Conklin
• 金额: $ 56.61万
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6898907
• 关键词: G protein; biological signal transduction; biotechnology; biotherapeutic agent; cardiac myocytes; cardiovascular pharmacology; cell growth regulation; chimeric proteins; drug design /synthesis /production; echocardiography; embryonic stem cell; fluorescence microscopy; gene targeting; genetic manipulation; genetically modified animals; green fluorescent proteins; hypoxia inducible factor 1; laboratory mouse; receptor binding; receptor coupling; receptor expression; tissue engineering; transfection /expression vector; tropomyosin; ubiquitin; vascular endothelium

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。虽然G蛋白偶联受体（GPCR）控制着各种各样的生理反应，但生物学家目前缺乏这样复杂的工具来利用体内的这些相同过程。随着组织工程领域的成熟，我们需要一种可激活的“开”和“关”开关，以便在治疗组织移植回患者体内后很长一段时间内对其进行控制。我们已经使用GPCR来开发仅由合成配体激活的受体（RASSL）。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。为了在体内控制GPCR信号传导，我们将开发一系列RASSL来激活每个主要的G蛋白途径（Gs，Gi，Gq）。每个RASSL也将与绿色荧光蛋白（GFP）融合，并将在关键调控位点发生改变，导致额外的RASSL对下调具有抗性或超敏感性。 具体目的2.为了验证RASSL信号转导可以调节转基因小鼠的生理反应的假设，将每个RASSL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。将通过短期（ECG）和长期（心脏重塑、心肌病）反应确定RASSL诱导的效应。 具体目标3。为了测试RASSL激活对生长和发育的影响，将检查具有靶向四个基因组基因座的RASSL的胚胎干（ES）细胞衍生的心肌细胞：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（缺血诱导的）和泛素连接酶E2B（普遍表达的）。 这些目标为组织工程提供了一个RASSL工具箱。将来，RASSLs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。这些目标也将使我们能够为希望在其他组织中使用RASSLs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一个RASSL工具箱，以选择性地激活各种组织中的任何主要GPCR途径。