Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene

Abstract

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Figure 1

In vivo selection results in significant increases in F cells in mice transplanted with γ-globin/MGMT lentiviral vector-transduced β-thalassemic BM cells. (A) The percentages of F cells in the peripheral blood of mice transplanted with the indicated γ-globin/MGMT vector-transduced β-thalassemic BM cells are shown before (10-12 weeks after transplantation) and 1 month after completing (30-32 weeks after transplantation) administration of 3 doses of BG/BCNU. F-cell percentages are also shown for control mice that received no drug treatment. (B) Percentages of F cells in control untreated mice and in treated responding mice before and after drug administration. Data represent the mean ± SEM. Statistically significant differences are indicated by the P values, as determined by the Student t test.

Figure 2

In vivo selection results in significant increases in HbF in mice transplanted with γ-globin/MGMT lentiviral vector transduced β-thalassemic BM cells. (A) Cellulose acetate gel electrophoresis of RBC lysates from individual mice transplanted with V5-Mp-MGMT vector-transduced β-thalassemic cells (top panel) or V5-EF1-MGMT vector-transduced β-thalassemic cells (bottom panel) and either not treated or administered drug treatment as indicated in the Figure 1 legend. Vertical lines have been inserted to indicate a repositioned gel lane. (B) HbF levels as determined by quantitative HPLC are shown for individual mice transplanted with V5-EF1-MGMT–transduced β-thalassemic cells and left untreated or after drug selection, as indicated. Data represent the mean ± SEM. Statistically significant differences are indicated by the P value, as determined by the Student t test.

Figure 3

Amelioration of the anemia and RBC morphologic abnormalities of β-thalassemia after in vivo selection of mice transplanted with γ-globin/MGMT lentiviral vector-transduced cells. (A) Hb levels in the indicated groups of control, untreated and drug-treated animals, both before and after drug administration. Values are also shown for mice transplanted with mock-transduced β-thalassemic cells at 32 weeks after transplantation. Data represent the mean plus or minus SEM. Statistically significant differences are indicated by the P values, as determined by the Student t test. (B) Wright-Giemsa–stained blood smears are shown for a representative treated and untreated animal transplanted with V5-EF1-MGMT-transduced β-thalassemic cells, as indicated. Photomicrographs were generated using an Olympus microscope (model BX60F-3; Tokyo, Japan) and an Olympus digital camera (model DP71), 100×/1.3 NA oil objective.

Figure 4

In vivo selection results in an increase in the amount of HbF per expressing RBCs. HbF per positive cell (pg/cell) is shown for individual mice transplanted with V5-EF1-MGMT vector–transduced β-thalassemic BM cells both before (■) and after (▨) drug administration.

Figure 5

Increased levels of vector-transduced HSCs after in vivo selection of mice transplanted with γ-globin/MGMT lentiviral vector-transduced cells. (A) Schematic diagram of the γ-globin/MGMT lentiviral vector showing the pertinent restriction enzyme sites used in the Southern blot analyses. (B) Southern blot analysis of genomic DNA, cut with BglII, from the BM of individual mice (indicated by mouse number) transplanted with V5-EF1-MGMT–transduced β-thalassemic BM cells. DNA size ladder is shown in the leftmost lane, whereas the vector plasmid DNA as a positive control is shown in the last lane (plasmid). Numbers below each lane represent the vector copy number as determined by densitometry, relative to the “Standard,” which is DNA from a K562 clone that contains a single copy of an integrated GFP lentiviral vector (1.0). Dilutions of this DNA with naive K562 DNA to establish samples with vector copy numbers of 0.5 and 0.25. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Southern blot analysis of genomic DNA, cut with EcoRI or XbaI as indicated, from the BM of individual mice (indicated by mouse number) transplanted with V5-EF1-MGMT-transduced β-thalassemic BM cells, as indicated. These enzymes cut once within the provirus; therefore, each band reflects a unique host genomic-vector junction fragment. Vertical lines have been inserted to indicate a repositioned gel lane.

Figure 6

Secondary transplantation demonstrates sustained, increased levels of F cells, HbF, and Hb. (A) The percentages of F cells in control, untreated, and treated V5-MSCV-MGMT animal groups (as indicated), both at baseline (1° Before) and after (1° After) drug treatment are shown. Also shown are the values in secondary transplantation recipients (2°) of BM from control, untreated, and drug-treated animals. The levels of F cells in the mice after treatment and in secondary recipients derived from them were statistically different from the baseline values before treatment as determined by the Student t test. P values are shown for each, compared with the baseline value. (B) The Hb levels in 1° and 2° control (untreated) and treated animal groups (as indicated) are shown. Also shown is the mean Hb level in animals transplanted with mock-transduced cells. Data represent the mean plus or minus SEM. (C) Top panel: cellulose acetate gel electrophoresis of RBC lysates from a drug-treated, V5-EF1-MGMT animal before (Pre) and after (Post) BG/BCNU administration and from 2° transplantation recipients derived from this primary mouse. The samples of the first 2 control lanes and the 2° transplantation lanes were run on one gel as indicated by the vertical lines. Pre and post samples in the 1° transplantation lanes were run on different gels at different times because samples could not be preserved intact for 20 weeks. Bottom panel: Hb levels of the primary animal before (Pre) and after (Post) drug treatment and of 2° transplantation recipients derived from the primary animal.

Figure 7

In vitro selection of transduced cells before transplantation results in increased engraftment of γ-globin–expressing cells and improved Hb levels. (A) The percentage of HbF-positive cells at 10 and 18 weeks after transplantation, as assessed by FACS, is shown for mice receiving transplantations of either untreated (n = 18) or drug-treated (n = 17) lineage-negative BM cells. P values of Student t test are shown. (B) The mean Hb levels of mice receiving transplantations of untreated or drug-treated BM cells are shown. As a control, the mean Hb level of all mice from both groups that had no γ-globin–expressing cells is shown. One-way analysis of variance with Bonferroni multiple comparison showed that Hb levels of treated and untreated groups significantly differed from the Hb level of mice that lacked γ-globin expression (P < .001 and P < .05, respectively). The difference between treated and untreated groups did not reach statistical significance. Data are expressed as mean ± SEM. (C) Vector copy number determination in animals receiving in vitro selected cells. Left panel: Southern blot analysis using BglII-digested BM DNA from the indicated animals. Left lanes indicate the copy number standards, as described in Figure 5 legend. Numbers indicate the VCN, as determined by densitometry, relative to the copy number standards. In addition to the hybridizing band of correct size, animal 50 also shows a second band that is smaller and probably represents vector containing a small deletion. Right panel: Southern blot analysis of BM DNA from the indicated animals, digested with the enzymes as labeled.