Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models

Abstract

Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.

Figures

Figure 1

Transplantation of w1.6W-transduced lin− wasnull cells for long-term experiments. (a) Western blot analysis of WASP expression in lysates of lin− wasnull cells either untransduced or transduced with the w1.6W vector at low or high MOI. As control, WASP expression in human PBMCs is depicted. Numbers indicate VCN. (b) Donor cell engraftment measured by real-time PCR in the indicated cell types, 12 months after transplantation of transduced or control lin− cells. Mean ± SD is depicted. Lin− wt n = 8, black bars; low MOI n = 11, light grey bars; lin− wasnull n = 4, empty bars. (c) Donor cell engraftment measured by flow cytometry analysis, 12 months after transplantation of transduced or control lin− cells. Mean ± SD is depicted. High MOI n = 17, dark grey bars; lin− wasnull n = 4, empty bars. MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; VCN, vector copy number; WASP, Wiskott–Aldrich Syndrome protein.

Figure 2

WASP expression in hematopoietic cells 12 months after gene therapy. (a) Analysis of WASP expression in BM CD45+ cells, and splenic CD11b+, B220+, CD8+, and CD4+ cells. Histograms report WASP expression in a representative wasnull (white), wt (black) and high MOI gene therapy treated mouse (gray). Numbers represent the percentage of WASP+ cells in the high MOI gene therapy treated mouse. (b,c) Cumulative analysis of the percentage of WASP+ cells in the indicated cell types within the low MOI gene therapy group (b, n = 21) or the high MOI gene therapy group (c, n = 16). Dots represent values from each single mouse. Bars indicate the mean value of the distribution. *P < 0.05, Student t-test. MOI, multiplicity of infection; WASP, Wiskott–Aldrich Syndrome protein.

Figure 3

Long-term restoration of T-cell function after gene therapy. Splenic T cells were purified by immunomagnetic beads, and stimulated with 2 µg/ml anti-CD3 mAbs for 48 hours. (a) Proliferation was measured by 3H-thymidine incorporation. wt n = 11; wasnull n = 15; high MOI n = 16; low MOI n = 23; lin− wasnull n = 13. Results are expressed by the stimulation index (SI), i.e., the ratio between cpm of stimulated and nonstimulated cells. (b–e) Cytokine levels were measured by BioPlex technology in conditioned supernatants. wt n = 12; wasnull n = 11; high MOI n = 16; low MOI n = 23; lin− wasnull n = 6. Dots represent the measurement performed in each single mouse. Bars represent the median value. *P < 0.05 as compared to wasnull group, Mann–Whitney test. cpm, counts per minute; MOI, multiplicity of infection.

Figure 4

Long-term correction of B-cell migration after gene therapy. B cells were purified from the spleen of gene therapy treated or control mice. Migration through 5-µm pore transwells was measured in response to medium only or medium supplemented with 1 µg/ml CXCL13. Results are expressed as the percentage of input cells that migrated to the lower well. Each dot represents a single mouse. Bars depict the mean value. n = 5 per group. *P < 0.05 as compared to the wasnull group, Student t-test.

Figure 5

Twelve-month follow-up of gene therapy treated mice. (a) Survival curve of mice belonging to the lin− wt (filled squares, n = 15), high MOI (filled circles, n = 20), low MOI (filled triangles, n = 48), and lin− wasnull (filled inverted triangles, n = 34) groups. The log-rank test failed to detect any statistically significant difference between the different groups. (b) Incidence of tumors in mice treated with gene therapy as compared to control groups. White bars represent the percentage of nonhematopoietic tumors, while black bars represent the percentage of host-derived hematopoietic tumors. Note that donor-derived hematopoietic tumors were never observed. Numbers represent the percentage of tumor incidence. MOI, multiplicity of infection.

Figure 6

Follow-up of mice recipient of secondary bone marrow transplantation. (a) Western blot analysis of WASP expression in lysates of 129-wasnull lin− cells either untransduced or transduced with one (WA 1X) or two (WA 2X) hits of the w1.6W vector. As control, WASP expression in 129-wt lin− cells is depicted. Numbers indicate VCN. (b) Engraftment of donor cells in sex-mismatched 129-wasnull mice, as determined by Y chromosome-specific real-time PCR. For primary transplantations (p BM), dots represents measurements performed in total bone marrow cells, and horizontal bars depict the median value of the distribution. n = 4–6 mice per group. Arrows highlight mice chosen as donors for secondary transplants. For secondary transplantations, diamonds represent median ± interquartile range in total bone marrow cells (s BM, black) and spleen (s spl, white). wt GFP group: n = 10; WA 1X group: n = 9; WA 2X group: n = 9; wasnull GFP group: n = 4. n.s. = not significant, Mann–Whitney test. (c) VCN in hematopoietic cells of mice recipient of either primary or secondary transplantation. VCN was determined by real-time PCR and normalized for the percentage of engraftment. For primary transplantations (p BM), dots represent measurements performed in total bone marrow cells, and horizontal bars depict the median value of the distribution. n = 4–5 mice per group. Arrows highlight mice chosen as donors for secondary transplants. For secondary transplantations, diamonds represent median ± interquartile range in total bone marrow cells (s BM, black) and spleen (s spl, white). wt GFP group: n = 10; WA 1X group: n = 9; WA 2X group: n = 8; wasnull GFP group: n = 4. *P < 0.05, n.s. = not significant, Mann–Whitney test. (d) Survival curves of mice receiving secondary bone marrow transplantation. For wt GFP (filled squares), n = 11; for WA 1X (filled triangles), n = 16; for WA 2X (filled circles), n = 16; for wasnull GFP (filled inverted triangle), n = 11. P < 0.05 for wt GFP group as compared to all the other groups, log-rank test. BM, bone marrow; VCN, vector copy number.