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Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis.
Blood. 2003 Feb 15; 101(4):1284-9.Blood

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+Show Affiliations

Department for Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University, Sweden.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12393514

Citation

Woods, Niels-Bjarne, et al. "Lentiviral Vector Transduction of NOD/SCID Repopulating Cells Results in Multiple Vector Integrations Per Transduced Cell: Risk of Insertional Mutagenesis." Blood, vol. 101, no. 4, 2003, pp. 1284-9.
Woods NB, Muessig A, Schmidt M, et al. Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood. 2003;101(4):1284-9.
Woods, N. B., Muessig, A., Schmidt, M., Flygare, J., Olsson, K., Salmon, P., Trono, D., von Kalle, C., & Karlsson, S. (2003). Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood, 101(4), 1284-9.
Woods NB, et al. Lentiviral Vector Transduction of NOD/SCID Repopulating Cells Results in Multiple Vector Integrations Per Transduced Cell: Risk of Insertional Mutagenesis. Blood. 2003 Feb 15;101(4):1284-9. PubMed PMID: 12393514.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. AU - Woods,Niels-Bjarne, AU - Muessig,Arne, AU - Schmidt,Manfred, AU - Flygare,Johan, AU - Olsson,Karin, AU - Salmon,Patrick, AU - Trono,Didier, AU - von Kalle,Christof, AU - Karlsson,Stefan, Y1 - 2002/10/17/ PY - 2002/10/24/pubmed PY - 2003/3/14/medline PY - 2002/10/24/entrez SP - 1284 EP - 9 JF - Blood JO - Blood VL - 101 IS - 4 N2 - 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. SN - 0006-4971 UR - https://www.unboundmedicine.com/medline/citation/12393514/Lentiviral_vector_transduction_of_NOD/SCID_repopulating_cells_results_in_multiple_vector_integrations_per_transduced_cell:_risk_of_insertional_mutagenesis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/2002-07-2238 DB - PRIME DP - Unbound Medicine ER -