Tissue engineering with a complete RASSL toolbox

J. David Gladstone Institutes, San Francisco CA

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Abstract

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.