Analysis of self-inactivating lentiviral vector integration sites and flanking gene expression in human peripheral blood progenitor cells after alkylator chemotherapy.
Abstract Hematotoxicity is a major and frequently dose-limiting side effect of chemotherapy. Retroviral methylguanine-DNA-methyltransferase (MGMT; EC 2.1.1.63) gene transfer to primitive hematopoietic progenitor cells (CD34(+) cells) might allow the application of high-dose alkylator chemotherapy with almost mild to absent myelosuppression. Because gammaretroviral vector integration was found in association with malignant or increased proliferation, novel lentiviral vectors with self-inactivating (SIN) capacity might display a safer option for future gene transfer studies. We assessed the influence of chemoselection on integration patterns in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-treated and untreated human CD34(+) cells transduced with an SIN lentiviral vector carrying the MGMT(P140K) transgene, using ligation-mediated PCR (LM-PCR) and next-generation sequencing. In addition, for the first time, the local influence of the lentiviral provirus on the expression of hit and flanking genes in human CD34(+) cells was analyzed at a clonal level. For each colony, the integration site was detected (LM-PCR) and analyzed (QuickMap), and the expression of hit and flanking genes was measured (quantitative RT-PCR). Analyses of both treated and untreated CD34(+) cells revealed preferential integration into genes. Integration patterns in BCNU-treated cells showed mild, but not significant, differences compared with those found in untreated CD34(+) cells. Most importantly, when analyzing the local influence of the provirus, we saw no significant deregulation of the integration-flanking genes. These findings demonstrate that SIN vector-mediated gene transfer might display a feasible and possibly safe option for MGMT(P140K)-mediated chemoprotection of CD34(+) cells.
Grund
N
N
Department of Experimental Surgery, University Medical Center Mannheim, University of Heidelberg, 68167 Mannheim, Germany.
Maier
P
P
Giordano
F A
FA
Appelt
J U
JU
Zucknick
M
M
Li
L
L
Wenz
F
F
Zeller
W J
WJ
Fruehauf
S
S
Allgayer
H
H
Laufs
S
S
D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use*; D001773:Blood Cells / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic; D019076:Transgenes
D018952:Antigens, CD34; D018906:Antineoplastic Agents, Alkylating