Repopulation of B-lymphocytes with restricted gene expression using haematopoietic stem cells engineered with lentiviral vectors

Abstract

B-lymphocytes play a key role in the pathogenesis of many immune-mediated diseases, such as autoimmune and atopic diseases. Therefore, targeting B-lymphocytes provides a rationale for refining strategies to treat such diseases for long-term clinical benefits and minimal side effects. In this study we describe a protocol for repopulating irradiated mice with B-lymphocytes engineered for restricted expression of transgenes using haematopoietic stem cells. A self-inactivating lentiviral vector, which encodes enhanced green fluorescence protein (EGFP) from the spleen focus-forming virus (SFFV) promoter, was used to generate new vectors that permit restricted EGFP expression in B-lymphocytes. To achieve this, the SFFV promoter was replaced with the B-lymphocyte-restricted CD19 promoter. Further, an immunoglobulin heavy chain enhancer (Emu) flanked by the associated matrix attachment regions (MARs) was inserted upstream of the CD19 promoter. Incorporation of the Emu-MAR elements upstream of the CD19 promoter resulted in enhanced, stable and selective transgene expression in human and murine B-cell lines. In addition, this modification permitted enhanced selective EGFP expression in B-lymphocytes in vivo in irradiated mice repopulated with transduced bone marrow haematopoietic stem cells (BMHSCs). The study provides evidence for the feasibility of targeting B-lymphocytes for therapeutic restoration of normal B-lymphocyte functions in patients with B-cell-related diseases.

Figure 1

A schematic diagram illustrating the structure of lentiviral vectors used in the study. The SFFV-EGFP vector encodes enhanced green fluorescence protein (EGFP) from the spleen focus-forming virus (SFFV) promoter. The Eμ-MAR-PGK-EGFP lentiviral vector encodes EGFP from phosphoglycerate kinase (PGK) promoter. The engineered CD19-EGFP and Eμ-MAR-CD19-EGFP lentiviral vectors both encode EGFP from the CD19 promoter. The Eμ flanked with matrix attachment regions (MAR) were incorporated upstream of the PGK and CD19 promoter. Viruses were produced by co-transfecting HEK-293T cells with the vectors together with the pCMVΔR8.2 packaging plasmid and pMD.G encoding the VSVG envelope. Abbreviations: ψ, Psi domain; LTR, long terminal repeat; RRE, rev-responsive element; WPRE, woodchuck posttranscriptional regulatory element.

Figure 2

Flow cytometric profiles of EGFP protein expression by a B-lymphocyte cell line (Ramos), a T-lymphocyte line (Jurkat) and a fibroblast cell line (HEK-293T) transduced with the lentiviral vectors SFFV-EGFP or Eμ-MAR-PGK-EGFP. Percent cells expressing EGFP were determined (indicated) by expression of green fluorescence and by side scatter (SSC).

Figure 3

Flow cytometric profiles of EGFP expression. The profiles depict percentages of human and murine B-cell lines (Ramos and LK-35) and T cells (Jurkat and BW5147) expressing EGFP after transduction with the SFFV-EGFP, CD19-EGFP or Eμ-MAR-CD19-EGFP lentiviral vectors. The results presented in the figure show data from one experiment. The experiment was repeated at least three times on different days and using different batches of lentiviruses and cells. The SFFV-EGFP vector induced EGFP expression in 21.7±2.9% (mean±s.e.m.) Ramos B cells, 20.0±1.5% LK-35 B cells, 58.3±7.3% Jurkat T cells and 56.7±4.9% BW5147 murine T-cell line. The CD19-EGFP induced EGFP expression in 16.7±3.4% Ramos B cells, 11.0±1.5% LK-35 B cells, 2.3±0.3 Jurkat T cells and 1.3±0.3 BW5147 cells. The Eμ-MAR-CD19-EGFP vector induced EGFP expression in 18.3±1.8% Ramos cells, 21.7±2.2% LK-35 cells, 3.3±0.7% Jurkat cells and 4.3±0.3% BW5147 cells. The Eμ-MAR elements enhanced the level of EGFP expression (mean fluorescence intensity; MFI) by 3–5 folds in the B-cell lines.

Figure 4

Immune reconstitution of lethally irradiated mice with transduced BMHSCs. Two groups of four, 8-week-old Balb/c mice each were studied. One group was irradiated with 800 cGy of γ-irradiation but not reconstituted with BMHSCs and used as control to determine the effectiveness of the irradiation. The second group was irradiated and transplanted intravenously with 2 × 10⁶ BMHSCs from 8-week-old Balb/c mice from the same colony. FACS analysis of peripheral blood was performed on both groups to determine numbers of B- and CD4⁺ T-lymphocytes before and after transplantation using FITC-conjugated anti-CD19 or anti-CD4 antibodies. (a) The proportion of B- and T-lymphocytes before irradiation are shown, (b) 1 week after irradiation of the control group, (c) 10 weeks after transplantation with BMHSCs. Before irradiation, 49.8±0.9% of blood nucleated cells within the lymphocyte gate were CD4⁺ T-lymphocytes and 22.3±0.5% were CD19⁺ B-lymphocytes. After irradiation and reconstitution of the second group with BMHSCs, the distribution of lymphocytes within the lymphocyte gate were 51.0±2.2% CD4⁺ T-lymphocytes and 22.8±1.4% CD19⁺ B-lymphocytes.

Figure 5

Expression of EGFP in lymphocytes in mice transplanted with lentiviral transduced BMHSCs. Mean and s.e.m. for FACS data on the analysis of lymphocytes expressing EGFP: (a) histograms representing per cent B-lymphocytes (grey columns) and non-B-lymphocytes (black columns) within the lymphocyte gates that expressed EGFP in the blood (left) and spleen (right) of recipient mice. (b) Per cent B- and non-B-lymphocytes within the EGFP-positive gate. (c) MFI of EGFP expression levels in B- and non-B-lymphocytes in the blood and spleen of mice recipients of HBMSCs transduced with lentiviral vectors SFFV-EGFP, CD19-EGFP or Eμ-MAR-CD19-EGFP.