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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL060664-15
Application #
8320325
Study Section
Special Emphasis Panel (ZEB1-OSR-D (M1))
Program Officer
Lundberg, Martha
Project Start
1998-07-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
15
Fiscal Year
2012
Total Cost
$480,338
Indirect Cost
$228,852
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
Miyaoka, Yuichiro; Chan, Amanda H; Judge, Luke M et al. (2014) Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11:291-3
Spencer, C Ian; Baba, Shiro; Nakamura, Kenta et al. (2014) Calcium transients closely reflect prolonged action potentials in iPSC models of inherited cardiac arrhythmia. Stem Cell Reports 3:269-81
Park, Jason S; Rhau, Benjamin; Hermann, Aynur et al. (2014) Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal. Proc Natl Acad Sci U S A 111:5896-901
Spindler, Matthew J; Burmeister, Brian T; Huang, Yu et al. (2013) AKAP13 Rho-GEF and PKD-binding domain deficient mice develop normally but have an abnormal response to *-adrenergic-induced cardiac hypertrophy. PLoS One 8:e62705
Kazakia, G J; Speer, D; Shanbhag, S et al. (2011) Mineral composition is altered by osteoblast expression of an engineered G(s)-coupled receptor. Calcif Tissue Int 89:10-20
Hsiao, Edward C; Boudignon, Benjamin M; Halloran, Bernard P et al. (2010) Gs G protein-coupled receptor signaling in osteoblasts elicits age-dependent effects on bone formation. J Bone Miner Res 25:584-93
Hsiao, Edward C; Millard, Susan M; Louie, Alyssa et al. (2010) Ligand-mediated activation of an engineered gs g protein-coupled receptor in osteoblasts increases trabecular bone formation. Mol Endocrinol 24:621-31
Nakamura, Kenta; Salomonis, Nathan; Tomoda, Kiichiro et al. (2009) G(i)-coupled GPCR signaling controls the formation and organization of human pluripotent colonies. PLoS One 4:e7780
Hsiao, Edward C; Yoshinaga, Yuko; Nguyen, Trieu D et al. (2008) Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter. PLoS One 3:e2532
Conklin, Bruce R; Hsiao, Edward C; Claeysen, Sylvie et al. (2008) Engineering GPCR signaling pathways with RASSLs. Nat Methods 5:673-8

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