Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells

Didier Trono, Romain Zufferey
American Society of Hematology, 2000
Journal paper

Abstract

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)

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https://infoscience.epfl.ch/record/53720?ln=en

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Didier Trono, Romain Zufferey
American Society of Hematology, 2000
Journal paper

Abstract

Official source

https://infoscience.epfl.ch/record/53720?ln=en

About this result

Related concepts (12)

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Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis

Didier Trono

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.

2003

Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin(-) c-kit(+) Sca1(+) primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% +/- 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42. 0% +/- 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 +/- 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 +/- 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP(+) lentivirus vector-transduced colonies revealed vector PCR(+) GFP(+) (42%), vector PCR(-) GFP(-) (46%), and vector PCR(+) GFP(-) (13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.

2000

Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors

Didier Trono, Romain Zufferey

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting). Receptor-mediated inactivation of gene transfer was overcome by competing the EGF receptors on the target cells with soluble EGF or by removing the displayed EGF domain from the surface of the vector particles by factor Xa cleavage of a specific protease substrate engineered into its tethering linker (protease targeting). Intravenous infusion of nontargeted HIV-1 vectors led to maximal luciferase activity in liver and spleen with moderate or minimal activity in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, intravenous EGF-displaying vectors were expressed maximally in spleen with very low level luciferase expression detectable in liver (EGF-receptor rich). Liver transduction by the EGF-displaying vector was restored by pretreating the animals with soluble EGF suggesting that these vectors are inversely targeted to spleen.

2001

2000