1991 — 1995 |
Conklin, Bruce R |
K11Activity Code Description: Undocumented code - click on the grant title for more information. |
Mutant G Proteins Reveal Signaling Pathways @ University of California San Francisco
This application for a physician scientist award proposes to train a physician specializing in internal medicine to become an independent investigator in molecular pharmacology. Training will include didactic course work, seminars, and research on the molecular mechanisms of G protein structure and function. The applicant has two years of prior laboratory experience, but has been absent from the laboratory setting for two years while completing his residency. He has no previous training in molecular biology. G proteins amplify extracellular signals and sort the flow of signals from multiple receptors to multiple effector systems. Although cDNA cloning has identified a large number of G proteins (Gs, Gt1,2, Gi1,2,3, Go, Gz) only Gt and Gs have been assigned a specific function. The overall goal of the present project is to identify individual G proteins responsible for mediating specific signaling pathways. This goal will be accomplished by expressing mutant G protein alpha chains in mammalian cells and later in transgenic mice. G protein-effector coupling will be studied in cells that express "dominant positive" mutant G protein that are constituitively active (mimic hormone stimulation of endogenous G proteins). G protein- receptor coupling will be studied by cells expressing "dominant negative" mutant G protein that prevent activation of endogenous G proteins by monopolizing a rate limiting step such as binding to the hormone receptor. Dominant positive and dominant negative G protein mutants will be used as tools to study the effects of turning on or off specific signaling pathways in cells and later in transgenic animals. Emphasis will be placed on the creation of transgenic animals with cardiac expression of mutant G proteins to study the effects of manipulating specific signaling pathways in the heart without the systemic effects of pharmacologic treatments. Signaling pathways to be examined will include: beta-adrenergic stimulation of adenylyl cyclase (cAMP), m2-muscarinic inhibition of adenylyl cyclase, as well as the alpha1-adrenergic and m1-muscarinic stimulation of phospholipase C (inositol phosphates and calcium release), and of phospholipase A2 (arachidonic acid release).
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0.943 |
1998 — 2002 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Collaborative R01--Roles of G Proteins in Cardiomyopathy @ J. David Gladstone Institutes
DESCRIPTION (Adapted from the applicant's abstract) The cardiac responses to stress and cardiomyopathy share a common feature: altered signaling through G protein-- coupled receptor (GPCR) pathways that regulate the homeostatic balance of signaling in the heart. Restoration of normal homeostasis in cardiomyopathy is the basis for medical therapies that target GPCRs, such as the beta-adrenergic receptor. The common theme of the collaborative studies is to genetically alter G protein signaling pathways in order to identify the fundamental processes by which GPCRs and G proteins influence the development and/or progression of cardiomyopathy. This individual R01 is focused on specific G protein signaling pathways. In the heart, signals derived from over 20 different GPCRs are communicated through five major G protein pathways. In this proposal, cardiac-targeted, constitutive activation of specific G protein pathways are used to define the in vivo responses of the five major G protein pathways. To gain tissue-specific, temporally regulated control of G protein signals, two powerful biological approaches are combined. First, the primary signaling component of each G protein (Galpha) can be constitutively activated (Galpha*) so that each of the five major G protein pathways (Galphas*, Galphai*, Galphaq*, Galphal2*, or Galphal3*) can be examined. Second, the tetracycline transactivator (tet) system allows inducible expression of these constitutively active forms of Galpha in the adult mouse heart, avoiding possible developmental effects. Analysis of the transgenic mice will include detailed physiologic measurements and the use of DNA arrays for measuring changes in gene expression. These studies will provide answers to questions, such as: Which G protein pathway will cause cardiomyopathy? How does each G protein pathway alter the homeostatic balance of the heart? How can G protein signals be used to restore the homeostatic balance and ameliorate the pathology of an experimentally induced cardiomyopathy? A comprehensive, focused study of G protein signaling in the heart is proposed with three specific aims: 1. To selectively activate each of the major G protein signaling pathways in the heart by conditionally expressing mutationally activated Galpha subunits in transgenic mouse hearts. 2. To determine the physiological and gene expression effects of each major G protein pathway in a normal adult mouse heart. 3. To selectively activate G protein pathways in the setting of two experimentally induced cardiomyopathy models where G protein signaling has been shown to be altered. (End of Abstract)
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0.943 |
1998 — 2002 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gi Signaling and Cardiomyopathy @ J. David Gladstone Institutes
DESCRIPTION (adapted from the applicant's abstract): Human dilated cardiomyopathy (DCM) is associated with increased Gi protein levels, increased Gi signaling, and auto-antibodies that activate signaling by GI- coupled receptors. The goal of this proposal is to test the hypothesis that Gi signaling can cause DCM. Control of Gi signaling has been achieved by expressing in the heart a Gi-coupled receptor that has been specifically designed to be a Receptor Activated Solely by a Synthetic Ligand, or RASSL. The first RASSL (R1) is based on a Gi-coupled, kappa- opioid receptor. R1 contains mutations that reduce affinity for natural peptide agonists and yet allow activation by the drug spiradoline. Cardiac-specific, conditional expression of R1 in transgenic mice is achieved with a tetracycline-controlled expression system utilizing the a-myosin heavy chain promoter. Activation of R1 signaling by spiradoline administration results in acute slowing of heart rate and complete atrioventricular block, which are known effects of Gi signaling. Preliminary studies show that prolonged signaling by R1 causes a lethal form of congestive heart failure with anasarca (up to 60% weight gain), contractile dysfunction, and the histopathological features of DCM. The Gi signaling-induced cardiomyopathy can be phenotypically reversed by suppressing R1 expression, creating the potential for studies of disease recovery as well as disease onset. Specific Aims are: (1) to determine if receptor- stimulated Gi signaling in the heart can cause the characteristic anatomical, physiological and histopathological changes of DCM using echocardiography, perfused hearts, isolated heart tissue strips, quantitative morphometric analysis, gene expression, and biochemical markers of cardiomyopathy; (2) to determine if the DCM is influenced by the spatial or temporal nature of the Gi signal, by altering the anatomical location of the Gi signal, inducing continuous Gi signaling with a mutationally activated form of Gi, and reducing continuous basal Gi signaling by expressing a new RASSL (R2) that has a lower susceptibility to endogenous peptide agonists; and (3) to determine if a mouse heart with Gi-induced DCM can regain normal function on a physiologic, histologic, and cellular level.
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0.943 |
2003 — 2007 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tissue Engineering With a Complete Rassl Toolbox @ J. David Gladstone Institutes
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.
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0.943 |
2004 — 2006 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genmapp: a Tool For Pathway Analysis of Genomic Data @ J. David Gladstone Institutes
[unreadable] DESCRIPTION (provided by applicant): One of the great current challenges in biology is to interpret large genomic datasets in the context of known biological pathways. The GenMAPP (Gene MicroArray Pathway Profiler) computer program provides a freely available tool for organizing, storing, analyzing, and sharing pathway information with gene expression data. The prototype program, called GenMAPP 1.0, can be used to analyze gene expression data on MAPPs representing biological pathways or any other functional grouping of genes. MAPPs are database files produced with the graphics tools in GenMAPP that depict the biological relationship between genes or gene products. GenMAPP also allows for automated analysis of gene expression data in the context of hundreds of known gene groupings using an auxiliary program, MAPPFinder, and the Gene Ontology database. Although GenMAPP 1.0 is a powerful analysis tool with hundreds of users, it has limitations that warrant major revisions to the software. Our specific aims are: [unreadable] [unreadable] (1) To develop and maintain GenMAPP 3.0 as a robust, free, open-source application that adds drawing and editing enhancements and integrates MAPPFinder functions. Documentation and online tutorials will focus on the four major user groups: DNA-microarray users, journal readers, pathway experts, and biology students. [unreadable] [unreadable] (2) To redesign the underlying gene database to integrate data from the major genomic databases, including SWlSS-PROT, GenBank, RefSeq, LocusLink, Unigene, Gene Ontology, model organism databases, and custom user-specific databases. The MasterUpdate and MasterExtract open source programs will be developed to automatically build and update the gene database on a regular basis. The modular design of the new gene database will allow rapid updates and integration of species-specific databases at www.GenMAPP.org. Users can easily add custom databases for any other organism through a graphical user interface within the GenMAPP program itself. [unreadable] [unreadable] (3) To expand the import/export capabilities of GenMAPP with XML data exchange modules and improved HTML features that will allow cross-platform viewing of MAPPs. By capturing pathway information in a digital format, GenMAPP will act as a catalyst for sharing pathway information and building public pathway databases. With the completion these aims, GenMAPP 3.0 will provide a program that is robust, simple to use, and available without charge to the entire biological community. [unreadable] [unreadable]
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0.943 |
2007 — 2010 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genmapp-Cs, a Dynamic Resource Pathway Analysis @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Pathway-oriented visualization of genomic information enables biologists to interpret data in the context of biological processes and systems. We developed GenMAPP (Gene Map Annotator and Pathway Profiler) as a free, open-source, stand-alone computer program for organizing, analyzing, and sharing genome-scale data in the context of biological pathways. This program is widely used for DMA microarray studies (>12,000 unique user registrations, >200 publications). Continuing demands of our users and the ever-increasing size and complexity of datasets now require a major revision of GenMAPP. We have formed key alliances with other open-source bioinformatics pathway projects whose efforts complement GenMAPP. We joined the Cytoscape (www.cytoscape.org) consortium as core developers so that we can build GenMAPP-CS using the advanced layout and visualization tools already available in Cytoscape. To facilitate pathway exchange, we are working closely with community-driven standards, (e.g. BioPAX and SBML) and several major public pathway databases (e.g., Reactome;www.reactome.org) to enhance pathway content and exchange. To implement this plan, we propose three specific aims. Specific Aim 1: To build GenMAPP-CS, a client-server version of GenMAPP, to provide a dynamic environment for visualizing and analyzing genomic data on biological pathways. GenMAPP-CS is being developed as an open-source, Java-based program to visualize and analyze datasets that exceed GenMAPP's current capabilities by 10-100-fold, while maintaining user interfaces and specific functions intuitive to biologists. Specific Aim 2: To dynamically integrate GenMAPP-CS with major gene and pathway databases for over 20 major model organisms. The new GenMAPP-CS architecture will allow us to integrate gene exon, single nucleotide polymorphism (SNP), and protein domain information with probe information at a scale that is impractical in GenMAPP 2.0. Specific Aim 3: To enable GenMAPP-CS to visualize and analyze genome-wide splicing, polymorphism, and interaction datasets. The challenge of analyzing these massive and complex datasets is a major force driving the development of GenMAPP-CS.
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0.943 |
2008 — 2012 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tissue Engineering With a Modular Rassl Toolbox @ J. David Gladstone Institutes
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.
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0.943 |
2009 — 2015 |
Bruneau, Benoit Gaetan (co-PI) [⬀] Conklin, Bruce R Srivastava, Deepak [⬀] Yamanaka, Shinya (co-PI) [⬀] |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Induced Pluripotent Stem Cells in the Understanding and Treatment of Heart Diseas @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Over 1 million Americans suffer acute myocardial infarctions each year in the US, and among the survivors, 5 million are afflicted with heart failure. In addition, defects in cell lineage determination or morphogenesis underlie congenital heart malformations, the most common human birth defect. Survivors of congenital heart disease, who number over 1 million in the US, also often suffer from heart failure. Unfortunately, the heart has little regenerative capacity after injury. The recent discovery of human induced pluripotent stem (IPS) cells has opened the door for novel approaches to human disease, including the development of human cellular models for disease mechanisms and drug discovery, along with the potential for autologous cell-based therapies. We propose to assemble a team of investigators at the Gladstone Institutes and Stanford University to develop and capitalize on the potential of IPS cells in the treatment and understanding of heart disease. Methods of IPS generation avoiding genomic integration of DNA are developing rapidly, but continue to require refinement before use of iPS cells in humans; this hurdle will be addressed in this application. As methods for generating IPS cells are improved the team will work together to more efficiently generate iPS-derived cardiac cells for future therapy, capitalizing on their expertise in chromatin remodeling and microRNA (miRNA) biology and G-protein coupled receptor signaling. The team will generate iPS cell lines with fluorescent markers for progressive stages of cardiac differentiation using bacterial artificial chromosome (BAC) strategies. We will also attempt to reprogram somatic cells directly into cardiac progenitors. Survival and engraftment of cells in vivo will be examined in rodents and in large animals through our partners at Stanford. Disease-specific iPS cells will be generated to reveal novel aspects of human progenitor cell biology. This multidisciplinary team will bring broad and critical expertise to the NHLBI Progenitor Cell Consortium in an effort to aggressively capitalize on the promise and potential of iPS cells for heart disease The interaction with the Stanford group within our Hub will synergize and leverage the specific strengths of each group of investigators on the focused effort related to iPS cells. The specific aims are: 1) To develop integration-free and efficient methods of human IPS cell generation for future cell-based therapies; 2) To develop efficient directed differentiation of human IPS cells and methods of direct reprogramming; 3). To develop methods to use IPS cell-derived cardiac progenitors in animal models of cardiovascular disease and 4). To use disease-specific IPS cells for discovery of human cardiac progenitor biology and cardiovascular disease mechanisms.
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0.943 |
2009 — 2014 |
Boyer, Laurie A (co-PI) [⬀] Bruneau, Benoit Gaetan [⬀] Conklin, Bruce R Pollard, Katherine Snowden (co-PI) [⬀] Srivastava, Deepak (co-PI) [⬀] Yamanaka, Shinya (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Epigenetic Landscape of Heart Development @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Congenital heart defects (CHDs) are among the most common and most devastating birth defects in humans. Networks of transcription factors regulate cardiac cell fate and morphogenesis, and dominant mutations in transcription factor genes lead to most instances of inherited CHD. The mechanisms underlying CHDs that result from disruption of these networks remain to be identified, but regulation of gene expression within a relatively narrow developmental window is clearly essential for normal cardiac morphogenesis. In addition to transcription factors, epigenetic regulation via histone modifications, chromatin remodeling, and non-coding RNAs have key roles in modulating gene expression programs. Elucidating on a genome scale the physical and functional interactions between transcription factors and epigenetic regulators will considerably enhance our understanding of the control of heart development and will have important implications for understanding the mechanistic basis of CHDs. We propose a project as part of the NHLBI Heart Development consortium to provide an integrated epigenetic landscape for heart development, with a focus on CHD-related genes. We propose three major aims. Aim 1: Define genome-wide occupancy maps of transcription factors with known roles in cardiac development and human disease, and epigenetic regulators of transcription, in differentiating cardiomyocytes. Aim 2: Define the global function of transcriptional and epigenetic regulation in heart development and congenital heart disease. We will examine the effect of loss of function of cardiac transcription factors on epigenetic regulation, and alterations in epigenetic regulation in disease-specific induced pluripotent cells from CHD patients. We will also evaluate the global role of histone modifications in mouse heart development. Aim 3; Integrate microRNA expression and function into the regulatory networks governing cardiac development. High-resolution occupancy maps from Aims 1 and 2 will be analyzed specifically for miRNA promoter occupancy and combined with quantitative sequencing of miRNAs in differentiating cardiomyocytes. We will study the function of highly altered miRNAs, specifically those that target disease-causing cardiac transcription factors. Our studies will yield an important and transformative epigenetic atlas of heart development, which will link for the first time transcriptional and epigenetic regulators in a comprehensive network that will illuminate mechanisms underlying CHDs. RELEVANCE (See instructions): The proposed project will for the first time allow a new understanding of the gene networks that underlie congenital heart disease. Congenital heart disease is the most serious childhood illness, affecting 1% of children, and leading to significant mortality and long-term illness. However the underlying causes of these diseases are not understood. Our project will link the so-called epigenetic regulators that control how genes are turned on or off, to congenital heart disease, bringing new important insights into these diseases.
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0.943 |
2010 — 2014 |
Conklin, Bruce R |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Consortia For High-Throughput-Enabled Structural Biology Partnerships (U01) @ University of California, San Francisco
anticancer research; base; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Biophysics; California; Cardiovascular Diseases; Cell Fate Control; Cells; Complex; design; Development; DNA; Embryo; embryonic stem cell; Enhancers; Event; Gene Expression; Genetic Transcription; Goals; Hormonal; Hospitals; Image; In Vitro; in vivo; Individual; induced pluripotent stem cell; Institutes; Laboratories; Malignant Neoplasms; Methodist Church; Methods; Molecular; Molecular Biology; Mutagenesis; mutant; Mutation; Physiological; pluripotency; Post-Translational Protein Processing; Principal Investigator; programs; Promotor (Genetics); protein complex; Protein Structure Initiative; Proteins; Regenerative Medicine; Research; Research Institute; research study; response; San Francisco; self-renewal; Signal Transduction; Somatic Cell; Stem cells; structural biology; Structure; three dimensional structure; tool; transcription factor; Transcriptional Regulation; Universities; Work; X-Ray Crystallography
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0.918 |
2011 — 2014 |
Conklin, Bruce R Healy, Kevin Edward [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Disease Specific Cardiac Tissue Models @ University of California Berkeley
DESCRIPTION (provided by applicant): Treatments for cardiovascular diseases are significant unmet needs in the global medical community. We propose to develop in vitro models of diseased cardiac tissues by using precisely controlled artificial matrix preparations. The principal objective of this project is to establish an in vitro model of human cardiac tissue based on the reconstitution of synthetic models of the human ventricular myocardium with populations of patient specific human induced pluripotent stem (hiPS) cell-derived cardiomyocytes (hiPS-CMs). For this application we have chosen to focus on a single patient-specific disease, long QT syndrome (LQTS), as a basis for proof-of-principle of our methodology and workflow. Prolongation of the QT interval, the electrical manifestation of cardiac ventricular repolarization, is a major cause of cardiac arrhythmias and sudden death. Thus a LQTS patient-specific physiologically functioning 3D model of heart tissue would be a significant advancement for understanding, studying, and developing new strategies for treating cardiac arrhythmias and other cardiovascular diseases. We propose the following specifics aims to generate a human cardiac 3D tissue model. Aim 1. To optimize a directed differentiation method to obtain a consistent high yield (>75%) population of human CMs derived from either healthy hiPS or hiPS cells harboring gene mutations of LQTS, a potentially lethal mutation. Aim 2. To fabricate precisely defined 3D filamentous matrices that organize the structure of healthy hiPS-CMs into a 3D in vitro model of the human cardiac tissue. To assess the functional behavior of the model by examining its electrical and mechanical activity. Aim 3. To organize the structure of LQTS-hiPS-CMs into a 3D in vitro model of the human myocardium. To assess the functional behavior of the diseased tissue model by examining its electrical and mechanical activity, and response to pharmacological agents. (End of Abstract)
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0.909 |
2013 — 2017 |
Conklin, Bruce R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cell Production Core @ J. David Gladstone Institutes
To achieve the goals of this PPG we will need to grow large quantities of cardiomyocytes (CMs) from mouse embryonic stem (ES) cell sources, for affinity-purification mass spectrometry (AP-MS). CM production will be scaled up from 6-well plates, to large tissue flasks and then suspension culture in a bioreactor. Although large-scale cell production is inefficient in a single lab, combining our efforts into a core laboratory will increase cell production so that the AP-MS can be achieved efficiently. SPECIFIC AIMS SUMMARY Specific Aim 1: To provide high-quality cell production capabilities for program research projects. The cell production core will enable research projects to produce large quantities of cardiac progenitors and CMs derived from engineered mouse ES cells. By expanding cells in suspension, we can attain cell production levels that are at least two orders of magnitude greater than on petri dishes. These cells will be provided to the Proteomics Core to analyze biochemical complexes that underlie cardiac differentiation. Specific Aim 2: To develop new protocols for affordable high volume CM production. Bioreactors have successfully been used to produce large amounts of mouse and human CMs in academic and industrial laboratories, but the main limitation is the cost of reagents. Typical bioreactor runs will use 100 liters of media and state-of-the-art protocols can cost >$80,000 per run. We will adapt our current protocols to bring the cost down by over 70%, allowing for the proposed experiments in a large numbers of cell lines.
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0.943 |
2016 — 2019 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Identifying Therapeutic Targets For Rna Splicing-Related Cardiomyopathy @ J. David Gladstone Institutes
? DESCRIPTION (provided by applicant) Dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in the western world. RBM20 (RNA binding motif protein 20) is a recently described cardiac-specific, RNA splicing protein that is mutated in 2-3% of DCM. RBM20 directly binds to the RNA transcripts of many cardiomyopathy-associated genes and ensures the production of cardiac-specific protein isoforms. Human studies revealed a striking, unexplained, recurring pattern of seven tightly clustered single amino acid DCM-associated substitutions near the RS domain of RBM20. The tight pattern of heterozygous mutations in DCM patients and biochemical studies of RBM20 binding proteins suggest that human DCM mutations could have dominant negative interfering effects on the RBM20 splicing complex. Our central hypothesis is that the RBM20 RS domain mutants cause dominant negative interference resulting in pathophysiological RNA splicing that are distinct from loss-of-function mutations. We plan to develop a series of isogenic human induced pluripotent stem cell (iPSC)-cardiomyocytes (iPS-CMs). We have made multiple single-base RBM20 mutations without antibiotic selection (scarless) in iPSCs. We can efficiently produce RBM20 RS domain mutant (R636S) iPS-CMs, and the cells display cellular pathology consistent with DCM. RNA-Seq studies of R636S mutant iPS-CMs reveal >360 alternative splicing events, including many that have been previously reported. Our aims are: Aim 1. To test the hypothesis that RBM20 DCM-causing point mutations have a dominant negative effect, by analyzing pathological changes in splicing by RNA-Seq. We will engineer the seven recurring human DCM mutations into isogenic iPSCs, as well as RBM20 null mutations. The RNA-Seq studies will focus on identifying the most pathological alternative splicing events that could explain the DCM pathology. Aim 2. To test the hypothesis that RBM20 RS domain mutation (R636S) has different RNA- and protein- binding, we will use FLAG and APEX epitope tagging respectively, to selectively identify binding partners. We will use FLAG-RBM20-RNA binding assays (CLIP-Seq) to determine the exact RBM20 RNA binding sites. FLAG- and APEX-APMS will identify putative binding partners and regulators of RBM20. Aim 3. To determine if the putative binding partners are essential for RBM20 splice regulation. We will conditionally silence the expression of putative binding partners. We will use CRISPRi to modulate the expression of each gene, to determine if each protein is a potential therapeutic target. With the completion of these aims, we will directly test our hypothesis that would explain the pattern of human RBM20 mutations, and provide a molecular mechanism for pathological splicing. We will have defined the molecular targets and protein partners of RBM20. Drugs that alter RBM20 activity could enhance cardiac health and repair, via a novel mechanism. These studies will provide a foundation for developing a human iPS-CM-based platform to develop new therapeutics.
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0.943 |
2017 — 2020 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Protein Quality Control, Cardiomyopathy, Cardiotoxicity and Human Isogenic Ipscs @ J. David Gladstone Institutes
Dilated cardiomyopathy (DCM) is often associated with accumulation of misfolded proteins thought to result from aberrant protein quality control. Chemotherapeutic drugs that target the proteasome (a key component of protein quality control) are associated with cardiotoxicity and heart failure. We hypothesize that the DCM- associated BAG3 protein regulates protein quality control in the heart and plays a critical role in cardiomyopathy and cardiotoxicity. BAG3 serves as a scaffold that binds and coordinates two classes of molecular chaperones: heat shock proteins, HSPBs and HSP70s. The BAG3 complex is stress-inducible and orchestrates protein folding, proteasomal degradation, and autophagy?all critical steps in protein quality control. In cardiac and skeletal muscle, this chaperone complex is localized in the Z-disk, where it is poised to regulate specific sarcomere client proteins. Mutations in BAG3 cause DCM and mutation-specific clinical phenotypes, suggesting a link with distinct cellular processes and disease pathways. We will test specific models of BAG3 function by engineering point mutations in BAG3 in isogenic human induced pluripotent stem cells (iPSCs) to produce cardiomyocytes (iPS-CMs). We have made significant progress in iPSC genome engineering to produce iPS-CMs and model human cardiac disease. We are also developing gene regulation tools based on CRISPR inhibition (CRISPRi) to rapidly silence genes to validate putative BAG3 interactions. Our aims provide a clear path to these goals. Aim 1: Identify the cellular processes involved in BAG3 cardiomyopathy in isogenic iPSC lines bearing disease-associated BAG3 mutations. We are making heterozygous and homozygous isogenic iPS- CMs that harbor clinically relevant mutations in the endogenous BAG3 locus. Aim 2: Directly define the role of BAG3-binding partners in the development of a cardiomyopathy phenotype in iPS-CMs by silencing candidate BAG3 interactors with CRISPRi. We hypothesize that specific BAG3 protein-binding partners contribute to the DCM phenotype. Aim 3: Determine if proteasome inhibitors and other chemotherapeutics cause cardiotoxicity in a manner dependent on specific components of the protein QC network. We hypothesize that altered function of the BAG3 chaperone complex leads to enhanced cardiotoxicity of proteasome inhibitors. We propose to comprehensively determine the mechanistic role of the BAG3 network in human cardiomyocytes and in DCM. A fundamental understanding of BAG3-mediated cardiac protein quality control will provide insights into disease mechanism, drug toxicity, and potential therapeutic options. Our studies lay the foundation for predictive genetic testing to understand genetic disease and avoid cardiotoxicity. We are hopeful that mechanistic insights will lead to treatments for cardiomyopathy and diseases of aberrant protein quality control.
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0.943 |
2017 — 2021 |
Conklin, Bruce R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Therapeutic Genome Editing to Treat Best Disease @ J. David Gladstone Institutes
PROJECT ABSTRACT Progressive retinal dystrophies, including retinitis pigmentosa and macular degeneration, are frequently caused by dominant negative mutations in which the disease could be cured by the silencing the mutant allele. Until recently, this task was impossible. However, the advent of CRISPR/Cas9 genome editing raises the exciting possibility of curing the disease by selectively inactivating the dominant disease-causing allele, while preserving the normal allele. As a proof-of-concept, we will focus on bestrophin, a protein encoded by the BEST1 gene that forms a calcium-activated chloride channel expressed in the retinal pigment epithelium (RPE). The most common BEST1-related disease is called ?Best disease? (BD), which is caused by >200 different dominant-negative protein coding mutations that result in defective chloride channel function, sub- retinal lipid accumulation, and macular atrophy. Induced pluripotent stem cells (iPSCs) from BD patients develop into RPE with disease phenotypes, such as abnormal chloride channel conductance and bestrophin mislocalization. DNA excision with dual cutting Cas9 is remarkably efficient, and by targeting Cas9 with guide RNAs (gRNA) to common polymorphisms on the same allele as (in cis with) the disease mutation, we propose to eliminate the disease protein. By targeting common polymorphisms, we hope to treat a majority of BD patients with just a few gRNA pairs. Although therapeutic editing for BD is promising, many daunting challenges remain. 1. How can we be confident that inactivation of the disease allele will cure the disease? 2. How do we efficiently identify polymorphisms in cis within a 20-30 kb genomic window that can be used for dual Cas9 excision of one allele, while leaving the other allele intact? (Fig. 5). 3. What are the ideal methods to introduce editing DNA/RNA/proteins into RPE for efficient and specific editing? 4. How do we assess the off- target editing for different SNPs and different methods of inserting Cas9 into cells? 5. Can we minimize off- target DNA damage using alternative forms of Cas9? We will systematically address each of these questions with a combination of bioinformatics, cell biology, and bioengineering with these aims: Aim 1. Determine the efficacy of allele-specific editing and the rescue of BD-associated RPE phenotypes using fluorescent reporter iPSCs Aim 2. Test allele-specific gRNAs for inactivation of disease alleles in RPE from 10 BD patients Aim 3. Determine the fidelity of the most robust allele-specific editing BD is a fertile testing ground for therapeutic editing, since RPE can readily be derived from iPSCs for in vitro studies, and is already the target of cell and gene therapy trials. Our studies also have larger implications: our methods are applicable to any dominant negative genetic disease where the selective removal of a single allele could be therapeutic. For instance, dominant negative disease of photoreceptors (e.g., RHO), auditory cells, nervous system, and muscle, are potential targets in the future.
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0.943 |
2019 — 2021 |
Conklin, Bruce R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core C: Genome Engineering Core @ J. David Gladstone Institutes
PROJECT SUMMARY/ABSTRACT CORE C ? GENOME ENGINEERING CORE Core C is focused on providing the most up-to-date genome engineering technology for studies of cardiac development. We will develop and adopt emerging technology for CRISPR/Cas9-mediated gene deletions, single-base changes, transgene insertions, and epigenetic remodeling. The PPG proposal utilizes advanced genome engineering techniques. We will specifically provide genome engineering services of human iPSCs to efficiently deliver engineered iPSCs for cardiac differentiation. The Genome Engineering Core will also provide epigenome editing methods, that modify the cell's genome without cutting DNA. The CRISPR- associated nuclease (Cas9) has been modified so that the nuclease is inactive in making ?dCas? that can now be used to carry and localize a wide variety of bioactive molecules to any location in the genome. We pioneered the use of CRISPRi that silences gene expression in iPSCs, and are involved in developing better methods that activate gene expression (CRISPRa). Our team is an established leader in genome engineering, and has made efforts to improve every aspect of genome editing in human iPSCs to benefit the PPG investigators in their efforts to unravel the molecular basis of congenital heart disease. In the last 5 years, we made >50 different genetically modified human iPSC lines with point mutations that exactly mimic the disease mutations, generate insertions/deletions (indels) for gene knockouts, or introduce endogenous gene tags for molecular studies of protein function (Miyaoka et al., 2014, Huebsch et al., 2015, Mandegar et al., 2016, Judge et al., 2017). The Genome Engineering Core will adopt the latest methods for genome engineering, such as the use of CRISPR/Cas9 RNP-protein complexes (RNP) to introduce insertion/deletions (indels), or delete discrete portions of genes to inactivate them in iPSCs as well as in iPSC-derived cardiomyocytes, since RNP-mediated genome editing is more efficient and accurate in our experience. In addition, the Genome Engineering Core will provide genome engineering services for the insertion of transgenes at endogenous loci, as well as develop new CRISPR methods for the PPG investigators to further investigate the cardiac interactome. The Genome Engineering Core will develop a pipeline to deliver high-quality engineered iPSCs to the PPG projects with continuously updated techniques, to answer vital questions in heart development.
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0.943 |
2021 |
Conklin, Bruce R |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
C9orf72 Frontotemporal Dementia (Ftd) and Amyotrophic Lateral Sclerosis(Als): Using Patient Cells and Crispr to Reveal Therapeutic Approaches @ J. David Gladstone Institutes
PROJECT SUMMARY/ABSTRACT A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases. Given that there are no effective treatments for FTD (an Alzheimer?s-related dementia) and ALS, novel therapeutic strategies are urgently needed. Targeting the C9orf72 gene itself by CRISPR/Cas9 gene editing may provide a curative intervention. However, we need to learn about the biology of the C9orf72 gene in order to employ gene editing strategies. This work proposes novel applications of CRISPR gene editing technology to edit or silence the pathogenic C9orf72 disease allele in FTD/ALS patient derived iPSC. With the completion of these aims, we will have systematically evaluated three complementary methods for silencing a deleterious repeat expansion in the C9orf72 gene: (1) bi-allelic excision of non-coding DNA harboring only the repeat expansion (Aim 1), (2) allele- specific excision of the mutant allele containing the repeat expansion (Aim 1), (3) regulatory region disruption to selectively silence the C9orf72 repeat expansion (Aim 3). We will examine the ability of these editing strategies to correct disease pathology in cell types relevant to disease ? human cortical and motor neurons. We have developed fast and robust methods to generate neurons from human induced pluripotent stem cells (iPSCs) derived from controls and patients. Analysis of edited control cell lines will allow us to screen for unanticipated effects of precise gene edits on normal cellular function and fitness. Our findings will not only advance our understanding of potential therapeutic approaches, but will also inform our understanding C9orf72 biology, including C9orf72 gene regulation and potential mechanisms of disease. This and our future studies will develop a pipeline for systematically evaluating editing strategies that are potentially curative.
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0.943 |