B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome

Abstract

Background: Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach.

Objective: Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration.

Methods: We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein.

Results: After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19(+)CD21(-)CD35(-) and CD21(low) B cells and a reduction in B cell-activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients.

Conclusions: We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.

Keywords: B cell; Wiskott-Aldrich syndrome; gene therapy; lentiviral vector; primary immunodeficiency.

Fig E1

WASp expression and transduction levels after GT. A, Histogram plots showing WASp expression measured by means of cytofluorimetric analysis in different hematopoietic cells of Pt4 before and 2 years after GT and an HD. Negative controls are shown as gray histograms. B and C, Transduction levels are shown in the graph as VCNs per genome evaluated by means of quantitative PCR at different time points after GT in B cells purified either from PB (Fig E1, B) or BM (Fig E1, C).

Fig 1

WASp expression in B cells after GT. WASp-expressing B cells in PB (A) and BM (B). Asterisks define the difference between pre-GT and post-GT samples. ***P ≤ .0005, **P ≤ .005, and *P < .05.

Fig 2

Expression of WASp among B-cell populations. A, WASp mean fluorescence intensity (MFI) in PB B-cell subsets of pediatric HDs (pHDs). Bars indicate means and SEMs. **P < .005 and *P < .05. B, WASp MFI in B-cell subsets of treated patients is reported in individual graphs, with the time of the analysis indicated as months after GT between parentheses. All MFI values are normalized to the MFI of the negative control stained only with secondary antibody.

Fig 3

B-cell differentiation and in vitro migratory ability of B cells after GT. A, Proportion of pre-B-II large (CD10^intCD20^int), pre-B-II small (CD10^intCD20⁻), and immature B cells (CD10^intCD20⁺) determined on CD34⁻CD19⁺ cells and calculated on total BM precursor B cells, excluding recirculating mature B cells (CD10⁻CD20⁺). B-E, Proportion of B-cell subsets in PB (gated on CD19⁺; HD <3 years, n = 26; HD 3-12 years, n = 20). Box plots show medians, 25th and 75th percentiles, minimums, and maximums. Asterisks above horizontal lines indicate significant differences between patients with WAS and HDs. Asterisks without lines define differences between pre-GT and post-GT samples. **P ≤ .005 and *P < .05. F-H,In vitro migration of CD20⁺ cells isolated from age-matched HDs (white bars, n = 10) and patients with WAS before (black bars) and after (gray bars) GT (n = 2, Pt2 and Pt3) in the presence of SDF-1α (+) or medium alone (−). Percentages of migrated CD19⁺ cells (Fig 3, F), transitional cells (Fig 3, G), and mature naive B cells (Fig 3, H) were determined by using flow cytometric analysis.

Fig 4

Normalization of phenotypic alterations of B cells from patients with WAS and BAFF levels after GT. A and B, CD21 and CD35 expression on CD19⁺ cells in patients with WAS before and after GT and in HDs (n = 16). C and D, CD21^low B cells in patients with WAS before and after GT and in HDs (n = 45). E, BAFF levels before and after GT and in HDs (n = 24). Asterisks above horizontal lines indicate significant differences between patients with WAS and HDs. Asterisks without lines define differences between pre-GT and post-GT samples. **P ≤ .005 and *P < .05.

Fig 5

Decreased autoantibody production in patients with WAS after GT. IgG reactivities against 123 antigens were tested by using an autoantigen array. P values resulting from paired t tests of differential expression between pre-GT and matched post-GT samples are shown in the left panel. Blue and green bars refer to autoantibodies with higher expression in pre-GT and post-GT samples, respectively. The vertical line indicates a P value of less than .05. Data are represented as a heat map (right panel), with values mean centered and colors scaled from −2 to +2 SD. The legend shows z scores.