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Development of Gene Therapy for Hematopoietic Stem Cells using Lentiviral Vectors

Woods, Niels-Bjarne LU (2002)
Abstract
Hematopoietic stem cells are an ideal target for genetic manipulation for the purpose of curing hematological disorders as they have the ability to develop into all blood lineages and to self-renew. In this study we demonstrate that lentiviral vectors, based on HIV-1, can efficiently transfer genes into human hematopoietic progenitor and stem cells as assessed following stem cell transplantation in immune compromised mice. High efficiency transduction of repopulating cells was achieved in both primary (48±23%, n=6) and secondary transplant recipients (64±13%). These results demonstrate the ability of lentiviral vectors to efficiently transduce human pluripotent candidate stem cells. Modifications to the vector design were performed to... (More)
Hematopoietic stem cells are an ideal target for genetic manipulation for the purpose of curing hematological disorders as they have the ability to develop into all blood lineages and to self-renew. In this study we demonstrate that lentiviral vectors, based on HIV-1, can efficiently transfer genes into human hematopoietic progenitor and stem cells as assessed following stem cell transplantation in immune compromised mice. High efficiency transduction of repopulating cells was achieved in both primary (48±23%, n=6) and secondary transplant recipients (64±13%). These results demonstrate the ability of lentiviral vectors to efficiently transduce human pluripotent candidate stem cells. Modifications to the vector design were performed to optimize the vector for high-level transgene expression in the progeny of the repopulating cells. Eight-fold higher expression levels were achieved in mice in both the lymphoid and myeloid progeny cells compared to the original lentiviral vectors. We also analyzed the vector copy number in the bone marrow cells by semi-quantitative PCR. To our surprise we found that on average there were multiple vector copies integrated per transduced cell (5.6±3.3 n=12). While the multiple vector copy integration into stem cells is efficient in terms of transduction and expression, it may increase the risk for insertional mutagenesis. Transduction of murine embryonic stem cells was performed to study transgene expression throughout in vitro differentiation to hematopoietic cells. In addition, we optimized the conditions for gene transfer into murine hematopoietic progenitors in order to be able to test hematopoietic stem cell gene therapy in animal models of human disease. These studies demonstrate the ability of lentiviral vectors to efficiently transduce human hematopoietic stem cells, and advance lentiviral vectors as a tool for the treatment of hematological disorders. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof, MD, PhD Humphries, R. Keith, Terry Fox Laboratory, Vancouver, Canada
organization
publishing date
type
Thesis
publication status
published
subject
keywords
embryonic stem cell, Hematopoietic stem cell, lentiviral vector, transduction efficiency, transgene expression, multiple vector copy integration, insertional mutagenesis, NOD/SCID mouse, cytogenetics, Genetics, extracellulära vätskor, extracellular fluids, cytogenetik, Genetik, Haematology, Hematologi
pages
140 pages
publisher
Margareta Ingloff (Secretary) Dept. of Molecular Medicine & Gene Therapy, BMC A12, SE-22184 Lund, Sweden, tel. +46 46 222-0575.,
defense location
Segerfalksalen auditorium, Wallenberg Neurocentrum, Sölvegatan 17, Lund, Sweden
defense date
2002-10-22 13:00
external identifiers
  • scopus:0036242306
ISBN
91-628-5337-2
language
English
LU publication?
yes
id
b4c7bdc1-3f05-489c-a2d7-65e9c4790c20 (old id 465036)
date added to LUP
2007-10-01 13:21:05
date last changed
2017-03-12 04:25:02
@phdthesis{b4c7bdc1-3f05-489c-a2d7-65e9c4790c20,
  abstract     = {Hematopoietic stem cells are an ideal target for genetic manipulation for the purpose of curing hematological disorders as they have the ability to develop into all blood lineages and to self-renew. In this study we demonstrate that lentiviral vectors, based on HIV-1, can efficiently transfer genes into human hematopoietic progenitor and stem cells as assessed following stem cell transplantation in immune compromised mice. High efficiency transduction of repopulating cells was achieved in both primary (48±23%, n=6) and secondary transplant recipients (64±13%). These results demonstrate the ability of lentiviral vectors to efficiently transduce human pluripotent candidate stem cells. Modifications to the vector design were performed to optimize the vector for high-level transgene expression in the progeny of the repopulating cells. Eight-fold higher expression levels were achieved in mice in both the lymphoid and myeloid progeny cells compared to the original lentiviral vectors. We also analyzed the vector copy number in the bone marrow cells by semi-quantitative PCR. To our surprise we found that on average there were multiple vector copies integrated per transduced cell (5.6±3.3 n=12). While the multiple vector copy integration into stem cells is efficient in terms of transduction and expression, it may increase the risk for insertional mutagenesis. Transduction of murine embryonic stem cells was performed to study transgene expression throughout in vitro differentiation to hematopoietic cells. In addition, we optimized the conditions for gene transfer into murine hematopoietic progenitors in order to be able to test hematopoietic stem cell gene therapy in animal models of human disease. These studies demonstrate the ability of lentiviral vectors to efficiently transduce human hematopoietic stem cells, and advance lentiviral vectors as a tool for the treatment of hematological disorders.},
  author       = {Woods, Niels-Bjarne},
  isbn         = {91-628-5337-2},
  keyword      = {embryonic stem cell,Hematopoietic stem cell,lentiviral vector,transduction efficiency,transgene expression,multiple vector copy integration,insertional mutagenesis,NOD/SCID mouse,cytogenetics,Genetics,extracellulära vätskor,extracellular fluids,cytogenetik,Genetik,Haematology,Hematologi},
  language     = {eng},
  pages        = {140},
  publisher    = {Margareta Ingloff (Secretary) Dept. of Molecular Medicine & Gene Therapy, BMC A12, SE-22184 Lund, Sweden, tel. +46 46 222-0575.,},
  school       = {Lund University},
  title        = {Development of Gene Therapy for Hematopoietic Stem Cells using Lentiviral Vectors},
  year         = {2002},
}