1997 — 1998 |
Leiden, Jeffrey M |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Cardiovascular Sciences Training Grant |
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1998 — 1999 |
Leiden, Jeffrey M |
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. |
Gene Therapy For Duchenne Muscular Dystrophy
Duchenne Muscular Dystrophy is a progressive and lethal X-linked myopathy caused by mutations and deletions in the Dystrophin gene located at Xp21. The treatment of DMD has been complicated by the need to induce dystrophin expression in a wide range of skeletal muscles and in the heart. Thus far, this has been impossible using cell-based therapies or chemical transfection approaches. Therefore, recent interest has focused on the use of viral-based somatic gene therapy approaches to program recombinant dystrophin expression in the skeletal and cardiac muscle of DMD patients. In the studies described in this application, we propose to use systemic delivery of replication-defective adenovirus vectors to program skeletal muscle- and cardiac-specific expression of a dystrophin mini gene in a large animal model of DMD. Adenovirus vectors were chosen for these studies because recent work from several groups including our own has demonstrated that they represent the only currently available viral delivery system that is capable of efficiently programming recombinant gene expression in a large percentage of non-replicating skeletal and cardiac muscle cells in vivo following systemic administration. Our proposed studies will make use of skeletal muscle- and cardiac-specific transcriptional regulatory elements previously cloned and characterized by our laboratory to restrict expression of the dystrophin gene to the appropriate muscle cell types. In order to circumvent technical difficulties associated with use of the 14 kb dystrophin cDNA, we will use a smaller, naturally-occurring deletion mutant of the dystrophin cDNA. The feasibility of treating DMD by the systemic administration of replication- defective adenoviruses will initially be assessed in xmd dogs, a canine model of muscular dystrophy that has been demonstrated by us to display genetic, histopathological and functional features that closely resemble those of the human disease. In the studies described in this proposal, we plan to (i) generate a series of recombinant, replication-defective adenoviruses containing either the firefly luciferase, bacterial lacZ, or human minidystrophin genes under the control of the cardiac and skeletal muscle-specific regulatory elements described above, (ii) optimize the method of administration of these vectors in neonatal dogs, (iii) administer dystrophin-expressing adenovirus vectors to xmd dogs, and (iv) assess the effects of this virus on disease progression and on skeletal and cardiac muscle function. In addition, we will carefully assess the safety and potential side effects of adenovirus administration in these animals. These studies will have direct relevance to the therapy of DMD. Additionally, they will have important implications for the treatment of a variety of other inherited myopathies.
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1 |
1998 — 2000 |
Leiden, Jeffrey M |
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. |
Gene Therapy For Serum Protein Deficiencies @ Harvard University (Medical School)
A number of important human diseases are caused by the inherited or acquired deficiency of a single serum protein. These include diabetes mellitus, pituitary dwarfism, hemophilias A an B and the erythropoietin (Epo) responsive anemias. Many of these diseases are currently treated by repeated intravenous or subcutaneous injections of purified or recombinant proteins. Such therapies although often life-saving are inconvenient and expensive. Moreover in some cases there is insufficient recombinant protein available to treat all patients prophylactically while in others intermittent administration of recombinant protein fails to ameliorate all of the side effects of the disease. These problems have stimulated interest in the development of novel gene transfer approaches that could be used for the treatment of acquired and inherited serum protein deficiencies. The long term goal of the studies described in this continuing application is the development of a muscle based somatic gene transfer approach that can be used to stably program physiologically regulated therapeutic levels of Epo in the circulation of patients with Epo responsive anemias. The method to be used is based on findings from the first three years of this grant which have demonstrated that genetically modified skeletal myocytes can be used to stably deliver physiological levels of Epo to the systemic circulation of mice and monkeys. In the studies described below the investigators propose to 1) develop novel adenoviral, adeno-associated viral and plasmid vectors for in vivo gene transfer of the Epo cDNA into skeletal muscle; 2) test the ability of these vectors to stably deliver physiological levels of Epo to the circulation of mice and monkeys following IM injection; 3) develop tetracycline-regulatable and skeletal muscle specific Epo expression vectors and test them in vitro and in vivo; and 4) test the hypothesis that hypoxia-inducible transcriptional regulatory elements from the Epo, LDH-A, and PGK genes can be used to construct hypoxia inducible Epo expression vectors that can be used to deliver physiologically regulated Epo to the circulation following IM injection. Taken together these studies should lay the foundation for successful human gene therapy trials for patients with Epo responsive anemias. They should also be relevant to the treatment of a number of other acquired and inherited serum protein deficiencies.
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1 |
1998 — 2000 |
Leiden, Jeffrey M |
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. |
Mechanisms of Dilated Cardiomyopathy in Creb A133
Dilated cardiomyopathy (DC) represents an important cause of cardiovascular morbidity and mortality and consumes a disproportionate share of medical resources in this country. Despite recent advances in the treatment of DC, this disorder has a poor prognosis with 5 year mortality rates of 20-50 percent. Progress in understanding the pathophysiology of DC and in devising new therapies for this disorder has been limited by our relative lack of understanding of the molecular pathophysiology of the disease and by the lack of a small animal model which closely resembles the anatomical, physiological, and clinical features of the human disease. We have recently shown that transgenic mice expressing a dominant-negative form of the CREB transcription factor (CREBA133) under the control of the cardiac-specific alpha-MHC promoter reproducibly develop DC that resembles many of the anatomical, physiological and clinical features of human DC. In the studies described in these 3 collaborative R01 applications we propose to use this new mouse model to better understand the molecular pathways by which CREB regulates cardiac myocyte homeostasis and how perturbations in these pathways produce DC. Specifically we will 1) elucidate the CREB-dependent signaling pathways that are required to maintain cardiac myocyte homeostasis and determine how these pathways are perturbed in the CREBA133 mice with DC, 2) determine the role of apoptosis in the CREBAl33 DC and test the hypothesis that the cardiomyopathic phenotype can be ameliorated by expression of anti-apoptotic genes in the heart, 3) study excitation-contraction coupling, contractility, and calcium homestasis in the CREBA133 cardiac myocytes, 4) understand the myofibrillar and SR defects underlying cardiac myocyte dysfunction in the CREBA133 mice, 5) study ventricular remodeling and LV-arterial coupling during the development of DC in the CREBA133 mice, and 6) determine the effects of exercise conditioning, gender, and different modes of inhibiting the renin angiotensin system on progression of DC in the CREBA133 mice. These studies represent the continuation of an established collaboration between molecular biologists (Leiden), cell physiologists (Moss) mouse and human physiologists (Lang, Spencer) and clinical cardiologists (Leiden, Lang, Spencer) the Universities of Chicago and Wisconsin. Taken together the results of this work should provide us with important new insights into the molecular mechanisms underlying human DC and CHF.
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1 |
1998 — 2000 |
Leiden, Jeffrey M |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Transcriptional Control of Human T Cell Receptor Genes
DESCRIPTION (Adapted from the applicant's abstract): The normal development and activation of T lymphocytes are required both to ensure appropriate host responses to viral and neoplastic pathogens, and to prevent autoimmune destruction of host tissues. During the first four years of this R01 the applicant used the TCR alpha and beta genes as model systems to identify and characterize the transcription factors involved in regulating T cell development and activation. These studies have allowed the identification of several novel transcription factor families that appear to play important roles in regulating T cell- specific gene expression. These include the Ets protooncogenes, the GATA zinc finger proteins, and the CREB/ATF family of basic-leucine zipper transcription factors. There are at least 5 Ets proteins expressed in T cells: Ets-1, Elf-1, Fli-1, Ets-2, and GABPalpha. The available evidence suggests that the lymphoid-restricted factor, Ets-1 plays an important role in regulating the expression of genes such as TCR alpha and beta in developing thymocytes and resting T cells. In contrast, Elf-1 appears to play an important role in regulating a set of activation-specific T cell genes including GM-CSF, IL-3, IL-2Ralpha, and HIV-2. The applicant's previous studies have shown that Elf-1 is regulated at at least 3 post-translational levels: (i) by activation- specific phosphorylation, which is required for its DNA binding activity, (ii) by cooperative binding with specific AP1 and NF- kappaB transcription factors, and (iii) by regulated interactions with the retinoblastoma (Rb) gene product or related pocket proteins. Thus, Elf-1 appears to represent a functional link between activation-specific gene expression and cell cycle progression in T cells. Similarly, there are at least 6 ATF/CREB proteins expressed in T cells. Previous studies have suggested important roles for these proteins in regulating the expression of the TCR alpha and beta genes, and in controlling the activation-specific expression of molecules such as the proliferating cell nuclear antigen (PCNA), which is essential for cell cycle progression following T cell activation. In the studies described in this application, it is proposed to use a combination of genetic and biochemical approaches to more precisely elucidate the roles of Ets-1, Elf-1 and CREB/ATF transcription factors in regulating T cell development and activation. Specifically it is planned to (i) elucidate the molecular basis of Elf-1 activation following T cell activation, (ii) produce targeted disruptions of Ets-1 and Elf-1 in mice and study their effects on T cell development and activation, and (iii) use transgenic mice overexpressing a dominant-negative form of the CREB transcription factor to better define the role of CREB/ATF proteins in regulating T cell development and activation. These studies should have important implications regarding our understanding of the role of Ets and CREB/ATF transcription factors in regulating both T cell development and function. In addition, they may help to shed light on the molecular mechanisms involved in coordinately regulating gene expression and cell cycle progression in response to receptor-mediated signaling events.
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1 |
1998 — 1999 |
Leiden, Jeffrey M |
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. |
Transcriptional Regulation of Cardiomyocyte Development
DESCRIPTION (adapted from the applicant's abstract): Much has been learned about the molecular mechanisms that regulate the differentiation of the skeletal muscle lineages during mammalian development. In contrast, relatively little is currently understood about molecular bases of cardiac myocyte differentiation. Recent studies have demonstrated that distinct transcriptional pathways regulate skeletal and cardiac- specific gene expression and differentiation. However, until recently, the identity of the important transcription factors that regulate cardiac-specific gene expression have remained unclear. Members of the GATA family of zinc finger transcription factors have been shown to play important roles in the differentiation of multiple hematopoietic lineages. Recent evidence from the applicant and others suggests an important role for a new member of this family, GATA-4, in the coordinate regulation of cardiac-specific gene expression during heart development. GATA-4 expression is restricted to the pre-cardiac mesoderm and folding heart tube in the early mouse embryo. Moreover, GATA-4 expression precedes that of the cardiac contractile proteins by 0.5-1 day during mouse embryogenesis. Most importantly, GATA-4 binds to the promoter-enhancer elements of multiple cardiac genes, including the cardiac troponin C and troponin T genes, the ANF gene, the myosin light chain 1 gene, and the alpha-MHC gene. In addition, the forced expression of GATA-4 can directly transactivate the expression of at least some of these cardiac- specific promoter-enhancers in non-muscle cells. Taken together, these studies are consistent with the hypothesis that GATA-4 is one of the important cardiac myocyte determining genes. The studies described in this application are intended to (i) map the important functional domains of the GATA-4 transcription factor, (ii) directly test the role of GATA-4 in the regulation of cardiac gene expression and cardiac myocyte differentiation by studying the effects of targeted disruptions of the GATA-4 gene on cardiac myocyte development in vitro and in vivo, and (iii) map the regions of GATA-4 that are necessary for cardiac myocyte differentiation and gene expression during the in vitro differentiation of ES cells into embryoid bodies.
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