Development of Gene Therapy for Hematopoietic Stem Cells using Lentiviral Vectors

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
• supervisor: [unknown] [unknown]
• opponent: Prof, MD, PhD Humphries, R. Keith, Terry Fox Laboratory, Vancouver, Canada
• organization: Division of Molecular Medicine and Gene Therapy
• publishing date: 2002
• type: Thesis
• publication status: published
• subject: Neurosciences
• 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
• defense location: Segerfalksalen auditorium, Wallenberg Neurocentrum, Sölvegatan 17, Lund, Sweden
• defense date: 2002-10-22 13:00:00
• external identifiers: scopus:0036242306
• ISBN: 91-628-5337-2
• language: English
• LU publication?: yes
• additional info: Article: IIsao Hamaguchi, Niels-Bjarne Woods, Ioannis Panagopoulos, Elisabet Andersson, Cecilia Fahlman, Hanna Mikkola, Romain Zufferey, Didier Trono, Stefan Karlsson: Lentiviral Vector Gene Expression During ES Cell Derived Hematopoietic Development In Vitro. J of Virol, Nov. 2000; Vol. 74(22) p. 10778-10784. Article: IIHanna Mikkola, Niels-Bjarne Woods, Marketa Sjögren, Hildur Helgadottir, Isao Hamaguchi, Sten-Eirik Jacobsen, Didier Trono, Stefan Karlsson: Lentiviral Gene Transfer in Murine Hematopoietic Stem Cells is Compromised by a Delay in Proviral Integration and Results in Transduction Mosaicism and Heterogeneous Gene Expression in Progeny Cells. J of Virol, Dec. 2000; Vol. 74(24) p.11911. Article: IIINiels-Bjarne Woods, Cecilia Fahlman, Hanna Mikkola, Isao Hamaguchi, Karin Olsson, Romain Zufferey, Didier Trono, Stefan Karlsson: Lentiviral Gene Transfer into Primary and Secondary NOD/SCID Repopulating Cells. Blood, Dec. 1 2000; Vol. 93(12) p.3725. Article: IVNiels-Bjarne Woods, Arne Muessig, Manfred Schmidt, Johan Flygare, Karin Olsson, Patrick Salmon, Didier Trono, Christof von Kalle, Stefan Karlsson: Lentiviral Vector Transduction of NOD/SCID Repopulating Cells Results in Multiple Vector Integrations per Transduced Cell: Risk of Insertional Mutagenesis. (Blood in press, 2002). Article: VNiels-Bjarne Woods, Johan Flygare, Hanna Mikkola, Eva Nilsson, Karin Olsson, Thomas Relander, Johan Richter, Patrick Salmon, Didier Trono, Stefan Karlsson. Optimal Lentiviral Vector Design for High-Level Transgene Expression in the Progeny of NOD/SCID repopulating cells. (manuscript, 2002). The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Molecular Medicine and Gene Therapy (0131000135), Division of Molecular Medicine and Gene Therapy (013022010)
• id: b4c7bdc1-3f05-489c-a2d7-65e9c4790c20 (old id 465036)
• date added to LUP: 2016-04-04 10:11:49
• date last changed: 2022-04-08 05:16:38