Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome

Abstract

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Graphical abstract

Figure 1.

Evolution of gene marking and protein expression after GT. (A) WAS gene marking. (B) WASp expression (% WASp⁺ cells) by flow cytometry in different subsets; showing flow cytometry example from a representative patient (patient 5). dg, diploid genome; PMN, polymorphonuclear; Pt, patient.

Figure 2.

Reconstitution of cellular immunity. (A) Infection scores before and after GT (scoring includes events during the last year of F/U; median, 7.6 years; range, 5.3-8.8 years). (B) Eczema score before and after GT (scoring includes events during the last year of F/U; median, 7.6 years; range, 5.3-8.8 years). (C) Reconstitution of T cells after GT (left: absolute values; right: relative to baseline). (D) Comparison of CD4⁺:CD8⁺ T-cell ratio before and after GT (at latest F/U). (E) Comparison of percentage of naive CD8⁺ T cells (at latest F/U). (F) Evaluation of T-cell proliferation in response to anti-CD3 by thymidine incorporation assay (at 2-3 years after GT) and (G) by carboxyfluorescein succinimidyl ester proliferation assay (at 2 years after GT), data not available for patient 5. The shaded area represents the median with interquartile ranges of healthy controls (n = 21). (H) Deep sequencing of TCR repertoire (patient 1, 2 years after GT; patient 2, 1 year after GT; patient 3, 1 year after GT). The frequency of the top 100 most abundant unique clonotypes is expressed as a percentage of the total number of sequences obtained. F/U, follow-up; HC, healthy control; ULN, upper limit of normal; Y, year.

Figure 3.

Reconstitution of humoral immunity. (A) Reconstitution of B cells after GT (left: absolute values; right: relative to baseline). (B) WASp expression in B-cell subsets (percentage of WASp⁺ cells) measured by flow cytometry (2 years after GT). (C) Percentage of unswitched memory B cells after GT. (D) Percentage of class-switched memory B cells after GT. (E) Percentage of MZ-like B cells after GT. (F) Evolution of IgM levels after GT. Asterisks (∗) indicate values after chemotherapy. (G) Isohemagglutinin titers (before and 4-5 years after GT).

Figure 4.

Platelet, monocyte, dendritic cell, and macrophage (myeloid) reconstitution. (A) Evolution of platelet levels after GT (left axis) and comparison to VCN in positive CFUs (left axis). (B) Comparison of mean platelet volume before and after GT (latest F/U). Statistics: Wilcoxon signed-rank test. (C) Comparison of platelet counts (≥18 months after GT) in relation to the number of transduced CD34⁺ cells infused on day 0. (D) Monocyte (CD14⁺) WASp expression (percentage WASp⁺ cells) by flow cytometry (latest F/U). (E) Evaluation of podosome formation by actin and vinculin colocalization by confocal imaging in stimulated CD14⁺ cells. (F) Evaluation of cytoskeletal function by determination of the percentage of podosome-forming cells in monocyte-derived dendritic cells (left axis) in comparison to the neutrophil VCN at 12 months (right axis). (G) Evaluation of messenger RNA expression of M1 and M2 markers by quantitative polymerase chain reaction of monocyte–derived macrophages before and after GT in patient 2 (9 months after GT) and patient 4 (3 months after GT). Graphs represent mean ± standard deviation of duplicates analyzed for each graph bar. MPV, mean platelet volume.

Figure 4.

Platelet, monocyte, dendritic cell, and macrophage (myeloid) reconstitution. (A) Evolution of platelet levels after GT (left axis) and comparison to VCN in positive CFUs (left axis). (B) Comparison of mean platelet volume before and after GT (latest F/U). Statistics: Wilcoxon signed-rank test. (C) Comparison of platelet counts (≥18 months after GT) in relation to the number of transduced CD34⁺ cells infused on day 0. (D) Monocyte (CD14⁺) WASp expression (percentage WASp⁺ cells) by flow cytometry (latest F/U). (E) Evaluation of podosome formation by actin and vinculin colocalization by confocal imaging in stimulated CD14⁺ cells. (F) Evaluation of cytoskeletal function by determination of the percentage of podosome-forming cells in monocyte-derived dendritic cells (left axis) in comparison to the neutrophil VCN at 12 months (right axis). (G) Evaluation of messenger RNA expression of M1 and M2 markers by quantitative polymerase chain reaction of monocyte–derived macrophages before and after GT in patient 2 (9 months after GT) and patient 4 (3 months after GT). Graphs represent mean ± standard deviation of duplicates analyzed for each graph bar. MPV, mean platelet volume.

Figure 5.

Autoimmunity. (A) Percentage of VH4-34–expressing B cells before (data includes other patients with WAS who did not undergo GT, data not available for patient 5) and after GT; showing flow cytometry example from a representative patient (patient 4). Statistics performed between HCs and pre-GT groups: Mann-Whitney U test. (B) Evolution of CD21^loCD38^lo B cells (%) after GT. (C) Evolution of Tregs (CD4⁺CD25^hiCD127^lo; left: percentage; right: absolute values) after GT; arrows represent onset of autoimmunity flares. (D) Treg (CD4⁺CD25^hiCD127^loFOXP3⁺) WASp expression (percentage WASp⁺ cells) after GT (2 years after GT). (E) Evolution of transitional B cells (left: percentage; right: absolute values) after GT. (F) Percentage of IL-10–producing B cells (Bregs) in various B-cell subsets in HCs, including transitional (CD19⁺CD24^hiCD38^hi), MZ-like (CD19⁺CD24⁺CD38⁻), and mature (CD19⁺CD24^intCD38^int) B cells. Statistics: Wilcoxon signed-rank test. (G) Percentage of Bregs in patients before and after GT (2 and 5 years after GT) with either absence (AI⁻) or presence (AI⁺) of after GT autoimmunity; showing flow cytometry example from a representative patient (patient 2). (H) Comparative levels of Bregs (left: percentage; right: absolute values) in patients with (AI⁺) and without (AI⁻) post-GT autoimmunity. Graphs represent median with interquartile range. Statistics: Mann-Whitney U test. (I) Percentage of Bregs within transitional B cells in patients before and after GT (2 and 5 years after GT). ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001. MFI, mean fluorescence intensity.

Figure 5.

Autoimmunity. (A) Percentage of VH4-34–expressing B cells before (data includes other patients with WAS who did not undergo GT, data not available for patient 5) and after GT; showing flow cytometry example from a representative patient (patient 4). Statistics performed between HCs and pre-GT groups: Mann-Whitney U test. (B) Evolution of CD21^loCD38^lo B cells (%) after GT. (C) Evolution of Tregs (CD4⁺CD25^hiCD127^lo; left: percentage; right: absolute values) after GT; arrows represent onset of autoimmunity flares. (D) Treg (CD4⁺CD25^hiCD127^loFOXP3⁺) WASp expression (percentage WASp⁺ cells) after GT (2 years after GT). (E) Evolution of transitional B cells (left: percentage; right: absolute values) after GT. (F) Percentage of IL-10–producing B cells (Bregs) in various B-cell subsets in HCs, including transitional (CD19⁺CD24^hiCD38^hi), MZ-like (CD19⁺CD24⁺CD38⁻), and mature (CD19⁺CD24^intCD38^int) B cells. Statistics: Wilcoxon signed-rank test. (G) Percentage of Bregs in patients before and after GT (2 and 5 years after GT) with either absence (AI⁻) or presence (AI⁺) of after GT autoimmunity; showing flow cytometry example from a representative patient (patient 2). (H) Comparative levels of Bregs (left: percentage; right: absolute values) in patients with (AI⁺) and without (AI⁻) post-GT autoimmunity. Graphs represent median with interquartile range. Statistics: Mann-Whitney U test. (I) Percentage of Bregs within transitional B cells in patients before and after GT (2 and 5 years after GT). ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001. MFI, mean fluorescence intensity.

Figure 6.

Insertion site and multipotency analysis. (A) Proportional contributions of gene-modified cell clones to the full population. Integration sites are named by the nearest gene (listed to the right). Gray indicates pooled low-abundance clones. (B) Longitudinal analysis of clonal diversity as measured using the Shannon index. Values are shown for each patient for each cell subset sampled (key below). (C) Longitudinal progression of pluripotency, measured using the multipotency index applied to the most abundant 100 clones, for which measurement is most reliable (described in detail in supplemental Data Analysis). The x- and y-axes both show the evaluable samples for each patients, and the colored tiles on the heat map show the extent of sharing between samples from the same patient. The diagonal from lower left to upper right shows the sharing of each sample with itself, which is 100% identical. The sharing between different time points is shown by the color code on the right.