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. 2007 Sep 15;110(6):1779-87.
doi: 10.1182/blood-2006-11-053710. Epub 2007 May 11.

Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

Claudia R Ball  1 , Ingo H Pilz, Manfred Schmidt, Sylvia Fessler, David A Williams, Christof von Kalle, Hanno Glimm
Affiliations

Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

Claudia R Ball et al. Blood. .

Abstract

Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

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Figures

Figure 1
Figure 1
Experimental design. Murine bone marrow cells were transduced with retroviral vector coexpressing MGMT P140K and eGFP and transplanted into syngeneic lethally irradiated primary recipient mice. Primary recipients received either 105 or 4 × 105 transduced BM cells. Cohorts of both groups were treated monthly with different dosages of BCNU and O6BG as indicated.
Figure 2
Figure 2
Efficient selection of MGMT P140K–expressing long-term hematopoiesis by BCNU/O6BG. The percentage of peripheral blood cells that expressed GFP was determined by FACS. Mice were treated monthly with BCNU/O6BG. Control mice underwent transplantation but did not receive the subsequent chemotherapy. (A) Efficient selection of MGMT P140K–expressing hematopoiesis in primary transplant recipient mice was observed only in those cohorts of mice that received 4 × 105 cells (squares) and monthly administrations of 20 μg/g O6BG and 5 μg/g BCNU (black symbols) (*P < .05). Triangles indicate 105 cells transplanted; squares, 4 × 105 cells transplanted; white, no O6BG/BCNU treatment; gray, 10 μg/g O6BG/2.5 μg/g BCNU; and black, 20 μg/g O6BG/5 μg/g BCNU; (n = 12 for each group). The higher dose of BCNU and O6BG allowed selection of marked hematopoiesis even after (B) secondary (n = 10, P < .05) and (C) tertiary (n = 11, P < .05) transplantation.
Figure 3
Figure 3
Multilineage differentiation of BCNU/O6BG-selected MGMT P140K–expressing long-term hematopoiesis. FACS analysis of peripheral blood cells from unselected control mice and mice that received a transplant of 4 × 105 cells before (4 weeks after transplantation) and after repetitive monthly injections of 5 μg/g BCNU and 20 μg/g O6BG (8, 12, 16, 20, 24, 28, and 32 weeks after transplantation, indicated by successive bars) was performed 23 to 25 days after each cycle of BCNU/O6BG treatment. The GFP+ hematopoiesis contributed to erythroid (TER119), monocytic (MAC1), granulocytic (GR1), B-lymphoid (B220), and T-lymphoid (CD3) lineages in unselected control animals as well as in BCNU/O6BG-treated mice even after serial transplantation. In primary recipient mice (A), the marked hematopoiesis produced fewer erythroid cells and monocytic cells accompanied by a relative increase in the proportion of the transduced T lymphocytes (*P < .01). In secondary recipients (B), no difference in the lineage contribution of the transduced hematopoiesis was observed with or without BCNU/O6BG treatment (n = 10). An increase in the proportion of marked T-lymphoid cells was detected again after tertiary transplantation in BCNU/O6BG-treated recipients (n = 11, *P < .05), whereas all other cell lineages remained unaffected (C). Standard error of the mean (± SEM) is shown for 11 to 12 individual mice per bar.
Figure 4
Figure 4
The number of clones contributing to marked hematopoiesis is not affected by BCNU/O6BG treatment. Representative LAM-PCR analysis of peripheral blood of mice at different time points after transplantation of 4 × 105 cells. Peripheral blood samples were analyzed before (4 weeks after transplantation) and with (A) or without (B) repetitive monthly BCNU/O6BG treatments (8, 12, 16, 20, 24, 28, and 32 weeks after transplantation). Multiple insertion sites were detected at all time points irrespective of whether the mice were treated with BCNU/O6BG. DNA (5 ng) was extracted from peripheral blood samples at each time point. M indicates a 100-bp DNA ladder; -c, negative control (5 ng DNA extracted from untransduced C57BL/6J bone marrow cells); and IC, internal control band.
Figure 5
Figure 5
Clonality of serially transplanted long-term hematopoiesis. Representative LAM-PCR analysis of peripheral blood. Multiple clones contributed to the long-term hematopoiesis of the primary (A), secondary (B), and tertiary recipient mouse (C), and the number of these clones was not reduced by BCNU/O6BG administration. M indicates 100-bp DNA ladder; -c, negative control (5 ng DNA extracted from untransduced C57BL/6J bone marrow cells); and IC, internal control band.
Figure 6
Figure 6
Tracking analysis of individual clones. The activity of individual clones over time in mice that underwent primary (1°), secondary (pair 2°a, 2°b), and tertiary (pair 3°a, 3°b) transplantation is shown. All mice that underwent tertiary transplantation received bone marrow from the corresponding secondary mouse 2°b not 2°a. Clone numbers refer to listed clones in Table 1. Dark squares indicate time points where the clone was detectable either by LAM-PCR and sequencing or by clone-specific tracking PCR. n.d. indicates not done.
Figure 7
Figure 7
Similar reconstitution of split bone marrow from individual mice in pairs of secondary transplant recipients. Peripheral blood samples from pairs of secondary mice that received a transplant of bone marrow from identical donors were analyzed by FACS at monthly intervals. Kinetics (A) and differentiation (B) of GFP+ blood cells were similar in pairs of secondary mice receiving bone marrow from the same donor. In panel A, time points in months are indicated by successive bars. In panel B, successive bars indicate the proportion of erythroid progenitors, granulocytes, macrophages, B lymphocytes, and T lymphocytes of the GFP+ blood cells 12 weeks after transplantation. Representative pairs of secondary mice with (mice 1 a,b, 2 a,b, 4 a,b) or without BCNU/O6BG treatment (mice 3 a,b, 5 a,b) are shown.
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