1985 |
Smithies, Oliver |
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. |
Dna Controlling the Synthesis of Human Hemoglobins F &A @ University of Wisconsin Madison
The work for year 4 of this research project proposes to complete the DNA sequencing of all of the DNA from well 5' to the capping site of the G gamma gene to well past the poly A addition site of the A gamma gene of the fetal globin genes. This will include all the DNA between the two genes to continue our attempts at isolating the DNA on the 3' side of the A gamma gene in order to be able to have the complete sequence for the whole of the fetal globin gene region from a single chromosome.
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0.939 |
1985 — 2005 |
Smithies, Oliver |
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. 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. |
Genetic Control of Protein Structure and Synthesis @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): A central problem in genetics is how substantial shifts in the overall gene expression programs of individual cells are achieved during development and in adult cells when they adapt homeostatically to a disturbance. As a framework against which to investigate this problem, we propose a "two-state" hypothesis, which we will test in relation to the complex homeostatic shifts in gene expression program that accompany the development and regression of left ventricular cardiac hypertrophy, an important cardiovascular risk factor independent of hypertension. The hypothesis states that adult cells (such as cardiac myocytes) when stressed sufficiently can switch without cell division to an alternative state in which the expression of many genes differ co-ordinately. [unreadable] [unreadable] Specific aim i) will test whether individual cardiac myocytes switch expression of representative genes coordinately between two states, and whether the degree of hypertrophy is mediated by the proportion of cells in the two states. [unreadable] [unreadable] Specific aim ii) will increase the stringency of the tests by using a much broader panel of discriminators with several categorically different ways of inducing hypertrophy, and will also test whether the hypertrophic cells replicate the expression pattern of normal cells at an earlier stage of development. [unreadable] [unreadable] Specific aim iii) will test the hypothesis that the expression programs of cardiac myocytes in the hypertrophic state can under appropriate circumstances switch back to the normal state in animals in vivo, or in tissue culture ex vivo. [unreadable] [unreadable] Our proposal is, to our knowledge, the first attempt to formalize how complex shifts in gene expression programs are achieved in cells responding to homeostatic challenges, but the value of the proposed experiments does not depend on the correctness of the two-state hypothesis since each specific aim also addresses a gap in current knowledge related to cardiac hypertrophy.
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1 |
1986 — 1990 |
Smithies, Oliver |
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. |
Targeted Correction of Faulty Human Beta-Globin Genes @ University of North Carolina Chapel Hill
Our goal is to evaluate the future potential of targeted gene modification with a view to its eventual use for the gene therapy of beta-thalassemia and sickle cell disease. Because targeted gene modification is such a novel approach to the genetic manipulation of mammalian genomes, much of the planned work will be directed towards fundamental aspects of the procedure. The specific objectives are to determine (i) what types of targeted modification of the human beta-globin gene can be achieved, (ii) what factors influence the efficiency of the procedure, and (iii) what conditions must be met to obtain correctly regulated and full level expression of a modified beta-globin gene. The experiments related to the types and efficiency of targeting will be primarily with cells in culture. The experiments related to expression will first be carried out in tissue culture. Later experiments will involve normal and thalassemice mice having their bone marrows repopulated by stem cells with a beta-globin locus modified by gene targeting. At the conclusion of the study it is expected that a plasmid will have been constructed and tested which is capable of introducing a gene into the beta-globin locus of hematopoietic stem cells from beta-thalassemia or sickle cell homozygotes in such a way that the resulting red cells function adequately, and that a comparable plasmid will have been shown to effectively cure thalassemic mice without having to use life-threatening techniques.
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1 |
1991 — 1994 |
Smithies, Oliver |
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. |
Targeted Correction of Faulty Beta-Globin Genes @ University of North Carolina Chapel Hill
The long term objective of the proposed work is to evaluate the future potential of making targeted gene modifications by homologous recombination for the somatic therapy of beta thalassemia and sickle cell anemia. Four specific aims have been selected which in combination should assist in the development of a gene targeting mode of therapy. Each specific aim also explores some aspect of gene manipulation by homologous recombination that either has intrinsic interest outside the field of gene therapy, or should yield benefits not only for hemoglobinopathies but also for other genetic diseases. (i) DNA constructs will be developed with the specific aim of targeting beta globin genes efficiently while still maintaining their normal expression. (ii) Gene targeting in embryonic stem cells will be used to make a mouse strain having the human delta and betas globin genes in place of the mouse adult beta globin genes with the specific aim of providing animals for in vivo test of constructs designed to correct human globin genes. (iii) with the specific aim of developing conditions for gene targeting in bone marrow stem cells, a mutant hypoxanthine phosphoribosyl transferase (HPRT) gene will be corrected in these stem cells. (iv) A procedure will be tested to give targeted cells a controlled proliferative advantage over unaltered cells, with the specific aim of ensuring that cells returned to a recipient after gene targeting can effectively compete in vivo with uncorrected resident cells.
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1 |
1992 — 2016 |
Smithies, Oliver |
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. |
Animal Models For Studying the Genetics of Hypertension @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Our long-term objective remains to unravel the genetic complexities of essential hypertension and its complications. Specific aim (i) will test the hypothesis that blood pressure is controlled by many genetic and environmental factors that individually have only small effects but which in some combinations are detrimental and in others beneficial. Mice having pair-wise combinations of high and low expressing variants at genetic loci (Agtr1a, Npr1, and Pparg) in three different physiological systems will be generated. Telemetric blood pressure monitoring and quantitative RT-PCR will determine the effects on these mice of dietary salt and fat, and of relevant drug treatments, and how homeostatic compensations in their heart, kidneys, adrenals, liver and adipose tissues are affected. Specific aim (ii) will test the hypothesis that the renin gene outside the kidney is important in blood pressure maintenance. Ren1c-/- mice (which cannot produce renin anywhere), and mice with renin transgenes, RenTgs (which produce renin at different constant levels only in the liver) will be mated, and their Ren1c-/-RenTg progeny will be used to determine whether extra-renal renin can regulate blood pressure. We will transplant non-renin-producing kidneys from Ren1c-/-RenTg mice into wildtype mice to determine whether the Ren1c gene in extra-renal tissues, but not in the kidney, can control blood pressure. Specific aim (iii) will test the hypothesis that, during hypertensive cardiac hypertrophy, myocytes assume one of two states: hypertrophic but not expressing fetal genes, or hypertrophic and expressing fetal genes. We will induce and reverse hypertension in mice expressing fluorescent indicators of hypertrophy-responsive genes, and assess expression of the indicator and other genes in individual cells to determine whether genes switch concordantly or randomly in individual cells, and whether switched cells can resume their former state. Together these several studies should contribute to a better understanding of how genetic factors affect blood pressure, its homeostasis, and the complications of hypertension.
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1 |
1995 — 2004 |
Smithies, Oliver |
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. |
Targeted Correction of Faulty Beta Globin Genes @ University of North Carolina Chapel Hill
Gene therapy would ideally correct a mutant gene precisely without causing changes elsewhere in the genome. Homologous recombination has this potential, but has not yet been successfully used in this context. During the last grant period, we demonstrated correction of a faulty HPRT gene by homologous recombination in clonogenic hematopoietic progenitor cells, and showed that a truncated erythropoietin receptor transgene introduced into the HPRT locus of ES cells can give hematopoietic stem cells (HSC) from the resulting mice an advantage over wild type cells in competitive bone marrow transplantation. Building on this work, we have chosen three aims directed towards correcting mutant human globin genes in mice, but equally applicable to other defects treatable via HSC correction. Specific aim (i1) will test whether ex vivo homologous recombination in HSC can correct mutant genes at (A) the HPRT and (B) the B globin locus. Specific aim (ii) will develop a transgene able to give a controllable in vivo transplantation advantage specifically to HSC when inserted at and site in the genome. Including but not limited to the globin locus. Specific aim (iii) will combine the targeting procedures developed in aim (i) with an advantage sequence developed under aim (ii) to attempt therapy in mice carrying a mutant human beta globin gene (B-O or B-S). Correcting a mutant gene in HSC by homologous recombination, and showing that the simultaneous co-introduction of an advantage transgene into HSC can facilitate their engraftment in an affected donor, would constitute substantial advances both for gene therapy in particular and for bone marrow transplantation in general.
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1 |
1997 — 2001 |
Smithies, Oliver |
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. |
Models For Studying the Genetics of Hypertension @ University of North Carolina Chapel Hill
The objective of this renewal application is to continue work begun during the prior funding period focused on the analysis of the role of various candidate genes in hypertension through the manipulation of their dosage in genetically engineered mice. This prior work involved using gene targeting to create mice with zero, one, two, three, and four copies of candidate genes targeted at their normal chromosomal locations and then assessing the impact of these defined alterations on hypertension in the mouse. The studies demonstrated that gene dosage determines the levels of expression of these genes which either directly impacted on blood pressure, or had blunted effects on blood pressure due to homeostatic adjustments mediated by other genetic elements. The hypothesis that was developed from these prior studies and which is to be tested in the proposed studies are that variations in the level of expression of a variety of genes will lead to blood pressure changes that may be a reflection of increased expression of the manipulated genes or may be blunted or completely missed depending on the homeostatic adjustments mediated by other elements. Dr. Smithies interprets these blunted changes as virtual blood pressure changes since homeostatic adjustments (i.e.) altered chronic expression by other non-manipulated genes sensing blood pressure changes) which can be demonstrated result in an absence of detectable blood pressure changes. This hypothesis and its implications for the genetics of hypertension will be tested by the applicant in five specific aims. The first four aims will investigate the effects on blood pressure of altered expression of f our candidate genes: 1) the renin 1C gene 2) the Agtr1A gene 3) the type A natriuretic peptide receptor gene 4) the natriuretic peptide "clearance" type receptor gene. The final specific aim involves the development of a path diagram displaying the interaction of these genetic determinants from Aims 1-4. This includes predictions and the testing of these predictions using crosses between the mice generated for these studies.
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1 |
2006 — 2010 |
Smithies, Oliver |
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. |
Bradykinin, Nitric Oxide and Mitochondrial Dna Damage in Diabetic Complications @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): [unreadable] In our previous work we have demonstrated that genetic factors controlling the production of bradykinin (BK) and nitric oxide (NO) influence greatly the development of renal complications in mice made diabetic with streptozotocin (STZ) or by the Akita diabetogenic C86Y mutation in Ins 2. We also showed that diabetic nephropathy and several indicators of senescence increase progressively in the order wildtype < bradykinin receptor B2 null < Akita diabetic < B2 receptor null Akita diabetic. 8-OHdG content, point mutations and deletions in mitochondrial (mt) DNA increased in the same progression, as did indicators of oxidative stress. We now propose three specific aims and the generation of two new mouse models to determine the interplay between genetic factors that influence BK action, the production of NO, and diabetes-related increases in mutations in mtDNA. Specific aim 1 will determine the effect on diabetic complications of eliminating both BK receptors throughout the body, or in a tissue or cell specific manner; the effects of reducing oxidative stress in these mice will also be determined. Specific aim 2 will investigate the relationship between glomerular damage and mtDNA mutations in eNOS -/- diabetic mice in which we have found that oxidative stress is paradoxically less than in eNOS +/+ diabetic mice. Specific aim 3 will test the hypothesis that increasing the frequency of mtDNA mutations by introducing a proof reading defect into mitochondrial DNA polymerase gamma will exacerbate the complications in Akita diabetic mice even though oxidative stress is not further increased over that due to the diabetes alone. [unreadable] [unreadable]
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1 |
2009 — 2010 |
Smithies, Oliver |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Renal Processing of Albumin @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Increased albumin excretion is an indicator of renal damage in a wide variety of conditions, including hypertension, diabetes and aging. Correctly understanding the pathogenesis of albuminuria is prerequisite for interpreting its significance in different conditions, and for designing appropriate therapies. One current view of how the separates small and large molecules assumes that the major physical barrier to macromolecules is the podocyte slit diaphragm (SD) with the glomerular basement membrane (GBM) acting as a coarse pre-filter. Another view ascribes glomerular selectivity to the presence in the GBM of pores of different sizes. In 2003, I proposed a gel permeation/diffusion hypothesis and computer simulation postulating that the GBM is the predominant source of the size selectivity of the kidney, because there is limited space in the GBM (a concentrated gel) into which macro-molecules can permeate. The glomerular filtration rate (GFR) clearly governs all trans-glomerular water transport, but calculations by me and others indicate that diffusion rather than filtration governs Specific Aim (i) will test the primary assumption of the permeation/diffusion hypothesis by directly determining with transmission electron microscopy whether nanoparticles and nanoparticle-tagged proteins of different sizes reach the concentrations in the GBM predicted by a well proven gel permeation equation. Initial experiments will be in vitro with purified GBM;later experiments will be in animals. To test the diffusion postulate we will evaluate the renal distribution of tagged proteins when the renal artery is temporarily clamped following a protocol comparable to that used by Ryan &Karnovsky (1976). Specific Aim (ii) will test the prediction that albuminuria will develop when GFR is substantially decreased. Our preliminary data show that in normal mice angiotensin converting enzyme and/or cyclooxygenase inhibitors in high dose cause temporary reversible albuminuria, exacerbated by a low salt diet. We will carry out experiments to determine the factors leading to this albuminuria, and whether it can be achieved by a decrease in GFR without damaging the GBM, podocytes or tubules. PUBLIC HEALTH RELEVANCE The occurrence of albumin in the urine of an individual (albuminuria) indicates that something is wrong with the kidney. Understanding what changes in the kidney lead to albuminuria is therefore essential for designing proper treatments. We have recently advanced a novel hypothesis that challenges current views on the causes of albuminuria, and are proposing experiments to test this hypothesis.
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0.988 |