Lentiviral vectors incorporating a human elongation factor 1alpha promoter for the treatment of canine leukocyte adhesion deficiency

Abstract

Canine leukocyte adhesion deficiency (CLAD) provides a unique large animal model for testing new therapeutic approaches for the treatment of children with leukocyte adhesion deficiency (LAD). In our CLAD model, we examined two different fragments of the human elongation factor 1alpha (EF1alpha) promoter (EF1alphaL, 1189 bp and EF1alphaS, 233 bp) driving the expression of canine CD18 in a self-inactivating (SIN) lentiviral vector. The EF1alphaS vector resulted in the highest levels of canine CD18 expression in CLAD CD34(+) cells in vitro. Subsequently, autologous CD34(+) bone marrow cells from four CLAD pups were transduced with the EF1alphaS vector and infused following a non-myeloablative dose of 200 cGy total-body irradiation. None of the CLAD pups achieved levels of circulating CD18(+) neutrophils sufficient to reverse the CLAD phenotype, and all four animals were euthanized because of infections within 9 weeks of treatment. These results indicate that the EF1alphaS promoter-driven CD18 expression in the context of a RRLSIN lentiviral vector does not lead to sufficient numbers of CD18(+) neutrophils in vivo to reverse the CLAD phenotype when used in a non-myeloablative transplant regimen in dogs.

Figure 1

Construction, and testing of lentiviral vectors. (a) Schematic of the vector constructs. EF1α, elongation factor 1α; MSCV, murine stem cell virus; cPPT, central polypurine tract; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; RRE, Rev-responsive element; RSV, Rous sarcoma virus. (b) Transduction efficiency in LAD EBV B-cells (ZJ). ZJ cells (2.5 × 10⁵/well) were incubated with increasing volumes of each vector (240x concentrated) in RetroNectin™ coated, non-tissue culture treated, 24-well plates at 37°C. Following transduction, cells were analyzed for CD18 expression by flow cytometry on Day 5. The volumes of each concentrated vector (μl/well) are shown on the x-axis. The percentage of CD18⁺ cells is indicated on the y-axis. (c) Comparison of CD18 expression in ZJ cells. Mean fluorescence intensities (MFIs) of the three vectors transduced at 0.01 μl/well are shown. The MFI of an untransduced control is shown for comparison.

Figure 2

Transduction of CLAD CD34⁺ cells with SIN lentiviral vectors expressing canine CD18. (a) CD34⁺ bone marrow cells (5 × 10⁵/well) from CLAD pups were added to RetroNectin™ coated, non-tissue culture treated, 24-well plates. StemSpan SFEM + 10% FBS, along with 5 μg/ml of protamine sulfate, and a cytokine cocktail consisting of 50 ng/ml each of canine IL-6, canine SCF, human Flt3-L, human TPO, and human G-CSF were also added per well. Vectors were added at MOIs of 10 and 100. Plates were spinnoculated (2500 rpm, 32°C, 30 min) and incubated at 37°C. Following transduction, cells were analyzed for CD18 expression by flow cytometry on Day 5. The x-axis indicates CD18 expression, while the y-axis indicates the side scatter (SSC). The percentages of CD18⁺ cells are shown in the upper right-hand corner of each dot plot. (b) The mean fluorescence intensity (MFI) of CD18 expression in CLAD CD34⁺ cells following transduction with the three vectors (Day 5). CD18⁺ cells were gated as indicated by the selected areas in Figure 2a. Each vector is shown as follows: EF1αL (purple), EF1αS (blue), and MSCV (orange). MFIs corresponding to an untransduced control (gray), and that of a normal dog (black) are shown for comparison.

Figure 3

(a) Genomic PCR from peripheral blood leukocytes for canine CD18 cDNA integration. Genomic DNA was isolated from peripheral blood leukocytes 8 weeks (3 weeks for dog LV4) following infusion of vector-transduced cells for the lentiviral vector (LV1-4) and at 12 weeks following infusion for the foamy viral vector (FV1-4) and the γ-retroviral vector (RV1,2). 100 ng of genomic DNA was used as a template to assess the integration of canine CD18 cDNA by PCR. Std, 100-bp size standard; CLAD, untreated CLAD dog. (b) Change in the percentage of CD18⁺ cells between Day 5 and Day 15 following transduction. CLAD CD34⁺ cells transduced with the previously stated cytokine cocktail in RetroNectin™ coated 24-well plates, analyzed for CD18 expression on Day 5 or after an additional 10 days in growth factors to differentiate the cells down the myeloid lineage (Day 15).