Drug selection with paclitaxel restores expression of linked IL-2 receptor gamma -chain and multidrug resistance (MDR1) transgenes in canine bone marrow

Abstract

Unstable expression of transferred genes is a major obstacle to successful gene therapy of hematopoietic diseases. We have investigated in a canine large-animal model whether expression of transduced genes can be recovered in vivo. Mixed-breed dogs had undergone autologous bone marrow transplantation (BMT) with stem cell factor and granulocyte-colony-stimulating factor-mobilized retrovirally marked hematopoietic cells. The bicistronic retroviral vector construct allowed for coexpression of MDR1 and human IL-2 receptor common gamma-chain cDNAs. The latter gene is deficient in X-linked severe combined immunodeficiency. After initial high-level expression, P-glycoprotein and the gamma-chain were undetectable in blood and bone marrow 17 months post-BMT. Six months later, one dog was treated i.v. with 125 mg/m2 paclitaxel. Three administrations restored expression of the two linked genes to high levels in blood and bone marrow. Two dogs treated with higher paclitaxel doses died from myelosuppression after the first administration. As determined by flow cytometry, both genes were expressed in granulocytes, monocytes, and lymphocytes of the surviving animal. PCR analysis of DNA from peripheral blood confirmed that the retroviral cDNA was increased after paclitaxel treatment, suggesting enrichment of transduced cells. P-glycoprotein was detectable for more than 1 year after cessation of paclitaxel. Repeated analyses of blood and bone marrow aspirates gave no indication of hematopoietic disturbance after BMT with transduced cells and paclitaxel treatment. In summary, we have shown that with the use of a drug-selectable marker gene, chemotherapy can select for cells that express an otherwise nonselected therapeutic gene in blood and bone marrow.

Figure 1

Retroviral vector in this study. HGMDR, Harvey murine sarcoma virus backbone; human IL2RG cDNA, IRES, human MDR1 cDNA. Selected restriction sites used for construction and analysis of HGMDR are indicated. Drawing is not to scale. LTR, long terminal repeat.

Figure 2

Expression of P-gp in canine peripheral blood. For detection of P-gp, peripheral blood cells were stained with mAb MRK16 or a nonbinding isotype control, followed by staining with an FITC-labeled anti-mouse IgG antibody. Histograms display expression of P-gp in peripheral blood cells of animal M862 at indicated time points. As a negative control, blood cells from two normal, untreated dogs were analyzed (Bottom).

Figure 3

Expression of P-gp in canine bone marrow. P-gp was analyzed by staining of mononuclear bone marrow cells with mAb MRK16 or a control antibody. Cells were then labeled with an FITC-labeled antibody to murine IgG, followed by flow cytometry. Shown is expression of P-gp in bone marrow at indicated time points. (Bottom Left) A normal control animal. (Bottom Right) Cells from a dog (M801) transplanted with the same retroviral vector are analyzed before chemotherapy with paclitaxel. This animal died after the first chemotherapy because of paclitaxel toxicity.

Figure 4

Peripheral white blood counts during paclitaxel treatment. Neutrophil and lymphocyte counts were taken before and after three paclitaxel treatments (125 mg/m²). Normal canine neutrophil count is 3,600–12,500 cells per μl, and lymphocyte count is 1,000–4,800 cells per μl.

Figure 5

Multilineage expression of P-gp. (A) Gating for granulocytes (R1), lymphocytes (R2), and monocytes (R3) in forward and side scatter plot. (B) Cells were stained for P-gp expression 28 months post-BMT. (C) Cells were stained for γc with a phycoerythrin-labeled anti-CD132 antibody 27 months post-BMT.

Figure 6

Expression of γc in blood. (Left) Peripheral blood cells from dog M862 were analyzed after three courses of paclitaxel treatment. (Right) Cells from an untreated, normal dog were stained with a phycoerythrin-labeled mAb to CD132 or a phycoerythrin-labeled control antibody.

Figure 7

PCR detection of the transgene in peripheral blood of the dog. A fragment of the bicistronic γc-IRES-MDR1 cDNA was amplified from 200 ng of DNA with primers specific for the γc (sense primer) and IRES (antisense primer). As a control, a fragment from the canine corticotrophin-releasing hormone gene was amplified under identical conditions. Shown are analyses from blood at the following time points: 17 months post-BMT (lane 1), 25 months (lane 2), 27 months (lane 3), 28 months (lane 4), and 43 months post-BMT (lane 5). As a negative control, blood from a normal, untreated animal is shown in lane 6.