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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-07-2238.
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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1284-1289
GENE THERAPY
Lentiviral vector transduction of NOD/SCID repopulating cells
results in multiple vector integrations per transduced cell: risk of
insertional mutagenesis
Niels-Bjarne Woods,
Arne Muessig,
Manfred Schmidt,
Johan Flygare,
Karin Olsson,
Patrick Salmon,
Didier Trono,
Christof von
Kalle, and
Stefan Karlsson
From the Department for Molecular Medicine and
Gene Therapy, Institute of Laboratory Medicine, Lund University, Lund,
Sweden; the Department I of Internal Medicine and
Institute for Molecular Medicine and Cell Research, University of
Freiburg, Freiburg, Germany; the Department of Genetics
and Microbiology, University of Geneva Medical School, Geneva,
Switzerland; the Program for Molecular and Gene Therapy, Division of
Experimental Hematology, Cincinnati Children's Hospital Research
Foundation, Cincinnati, OH.
Efficient vector transduction of hematopoietic stem cells is a
requirement for successful gene therapy of hematologic disorders. We
asked whether human umbilical cord blood
CD34+CD38lo nonobese diabetic/severe
combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be
efficiently transduced using lentiviral vectors, with a particular
focus on the average number of vector copies integrating into these
primitive progenitor cells. Mouse bone marrow was analyzed by
fluorescence-activated cell-sorter scanner and by
semiquantitative polymerase chain reaction (PCR) to determine
the transduction efficiency into SRCs. Lentiviral vector transduction
resulted in an average of 22% (range, 3%-90%) of the human cells
expressing green fluorescent protein (GFP), however, multiple
vector copies were present in human hematopoietic cells, with an
average of 5.6 ± 3.3 (n = 12) copies per transduced cell. To
confirm the ability of lentiviral vectors to integrate multiple
vector copies into SRCs, linear amplification mediated (LAM)-PCR was
used to analyze the integration site profile of a selected mouse
showing low-level engraftment and virtually all human cells expressing
GFP. Individually picked granulocyte macrophage colony-forming
unit colonies derived from the bone marrow of this mouse were
analyzed and shown to have the same 5 vector integrants within each
colony. Interestingly, one integration site of the 5 that were
sequenced in this mouse was located in a known tumor-suppressor gene,
BRCA1. Therefore, these findings demonstrate the ability of
lentiviral vectors to transduce multiple copies into a subset of
NOD/SCID repopulating cells. While this is efficient in terms of
transduction and transgene expression, it may increase the risk of
insertional mutagenesis.
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