Lentiviral vector integration sites in human NOD/SCID repopulating cells.
BACKGROUND
Recent observations of insertional mutagenesis in preclinical and clinical settings emphasize the relevance of investigating comprehensively the spectrum of integration sites targeted by specific vectors.


METHODS
We followed the engraftment of lentivirally transduced human cord blood (CB) progenitor cells after transplantation into NOD/SCID mice using a self-inactivating HIV-1-derived vector expressing the enhanced green fluorescent protein (EGFP).


RESULTS
The mean of transduction of CD34(+) CB cells was 41%, as deduced from the percentage of EGFP(+) cells before transplantation. At 3 weeks post-transplantation, the average of EGFP(+) cells in the human cell population was 65 +/- 8%, and increased to 75 +/- 10% at 12 weeks post-transplantation. In order to determine the proviral integration sites in human NOD/SCID repopulating cells (SRCs) we used the ligation-mediated polymerase chain reaction (LM-PCR) technique. Sixty-eight percent of the integrations were found to be located in RefSeq genes, most of them in intron regions. Twenty percent of these integrations occurred within a distance of 10 kb from the transcription start site; a percentage that is significantly lower compared to that observed in cells transduced by gammaretroviral vectors. Sixty-two percent of integrations occurred in genes with a biological function in cell metabolism, and four integrations were located in genes with a role in tumorigenesis.


CONCLUSIONS
These investigations indicate that integration of lentiviral vectors in human repopulating cells capable of engrafting NOD/SCID mice preferentially occur in coding regions of the human genome. Nevertheless, the clustering of integrations at the transcriptional start is not as high as that observed for gammaretroviral vectors.
Laufs
Stephanie
S
Research Program Innovative Cancer Diagnostics and Therapy, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. s.laufs@dkfz.de
Guenechea
Guillermo
G
Gonzalez-Murillo
Africa
A
Zsuzsanna Nagy
K
K
Luz Lozano
M
M
del Val
Coral
C
Jonnakuty
Sunitha
S
Hotz-Wagenblatt
Agnes
A
Jens Zeller
W
W
Bueren
Juan A
JA
Fruehauf
Stefan
S
D000818:Animals; D002478:Cells, Cultured; D002877:Chromosomes, Human / Q000378:metabolism; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016254:Mutagenesis, Insertional / Q000379:methods; D014161:Transduction, Genetic / Q000379:methods*; D016662:Virus Integration / Q000235:genetics*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins