title: Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis.
abstract: 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(+)CD38(lo) 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.
authors.affiliation: Department for Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University, Sweden.
authors.forename: Niels-Bjarne
authors.initials: NB
authors.lastname: Woods
authors.forename: Arne
authors.initials: A
authors.lastname: Muessig
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Johan
authors.initials: J
authors.lastname: Flygare
authors.forename: Karin
authors.initials: K
authors.lastname: Olsson
authors.forename: Patrick
authors.initials: P
authors.lastname: Salmon
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Stefan
authors.initials: S
authors.lastname: Karlsson
mesh_terms: D036541:ADP-ribosyl Cyclase / Q000032:analysis; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells; D003114:Colony-Forming Units Assay; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D016147:Genes, Tumor Suppressor; D005822:Genetic Vectors; D006098:Granulocytes; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008264:Macrophages; D008562:Membrane Glycoproteins; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D014162:Transfection; D016662:Virus Integration
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1