In vivo selection of MGMT(P140K) lentivirus–transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning
Steven P. Zielske, … , Jon R. Donze, Stanton L. Gerson
Steven P. Zielske, … , Jon R. Donze, Stanton L. Gerson
Published November 15, 2003
Citation Information: J Clin Invest. 2003;112(10):1561-1570. https://doi.org/10.1172/JCI17922.
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Article Genetics

In vivo selection of MGMT(P140K) lentivirus–transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning

Abstract

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU–conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus–transduced human SRCs following BG/BCNU treatment.

Authors

Steven P. Zielske, Jane S. Reese, Karen T. Lingas, Jon R. Donze, Stanton L. Gerson

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Figure 7

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Repopulation analysis. Genome and MGMT copies were determined in individ...
Repopulation analysis. Genome and MGMT copies were determined in individual CFUs as described in Methods and plotted as shown. (a) CFUs from a mixture of two K562 clones containing integrated provirus were analyzed by real-time PCR, and the numbers of MGMT copies versus genome copies were plotted for each sample. Solid lines indicate linear regression, and dashed lines are the 95% confidence intervals. (b) Data from CFUs obtained from CD34+ mass cultures and before transplant show scattered distribution of data points. (c) Data from human CFUs obtained from four twice-treated mice. Two lines were distinguishable in mice nos. 21 and 22, indicating CFU populations clustering around two different copy-number distributions. Data from mouse no. 23 were insufficient to discern multiple lines, and mouse no. 24 gave a single line, probably indicating a monoclonal population. Triangles represent points not included in the linear regressions. Regressions were obtained by separately combining data points represented using circles or squares. One to 4 points are off scale in the in vitro, mouse no. 21, and mouse no. 22 plots and were not included in the analysis.