Development of hemopoietic stem cell gene therapy for Niemann-Pick disease using the acid sphingomyelinase knock-out mouse model.
Item
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Title
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Development of hemopoietic stem cell gene therapy for Niemann-Pick disease using the acid sphingomyelinase knock-out mouse model.
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Identifier
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AAI9917648
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identifier
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9917648
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Creator
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Erlich, Shai Shmuel.
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Contributor
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Adviser: Edward H. Schuchman
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Date
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1999
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics | Health Sciences, Immunology | Biology, Cell
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Abstract
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Currently, no therapy is available for Types A and B Niemann-Pick disease. The overall goal of my research project was to develop hemopoietic stem cell gene therapy for Types A and B NPD using the ASM knockout mouse (ASMKO) as a model system. In order to achieve our goal, several specific aims have been established:;Characterization of the ASMKO mouse phenotype. Homozygous knock-out and heterozygous mice were analyzed and compared to normal animals on three levels: (A) Biochemical analysis; (B) Pathological analysis; (C) Clinical analysis. The baseline data obtained from these studies was used later to determine the success of the gene therapy and BMT experiments.;BMT into ASMKO mice. In order to provide the rationale for the development of hemopoietic stem cell gene therapy for NPD, we first transplanted normal bone marrow cells into newborn and adult ASMKO mice. A group of animals was transplanted and successfully engrafted animals were analyzed biochemically, pathologically and clinically. It was found that the extra-neuropathic disease phenotype can be treated successfully by BMT.;Transplantation of genetically corrected hemopoietic cells into ASMKO mice. Affected mice were also transplanted with retrovirally transduced bone marrow cells. Higher ASM activities could be achieved in these animals compared to the normal allogenic BMT results. Animals showing high engraftment will be further studied biochemically, pathologically, and clinically.;Development of a fluorescence-based selection system for the isolation of retrovirally transduced, metabolically-corrected hemopoietic stem cells. A selection method was developed which allows us to select the metabolically corrected hemopoietic cells following retrovirus transduction of the ASM gene into the ASMKO mouse bone marrow. The method utilizes an artificial, fluorescent Sphingomyelin substrate that cannot be metabolized in the absence of the enzyme. By combining this labeling method with antibody selection (based on cell surface markers), we were able to select the corrected cells from various bone marrow sub-populations, including the corrected stem and progenitor cell populations. These cells can now be used for in vivo transplantation of ASMKO mice. In addition, this selection system has proved to be useful as an assay for the in vitro assessment of gene transfer into hemopoietic stem cells.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
