Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome

Abstract

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Conflict of interest statement

A.J.T. holds equity in Orchard Therapeutics. A. Galy and S.A. work at Genethon (sponsor). All other authors declare no competing interests.

© 2022. The Author(s).

Figures

Fig. 1. Immune reconstitution after gene therapy.

a, Measurement of the total CD3+ T cell, CD19+ B cell, CD56+ natural killer (NK) cell and CD15+ neutrophil counts in blood at regular time points after gene therapy. Age-matched reference values are indicated by the gray lines (solid line, lower value; dashed line, upper value). b, Measurement of the VCN per cell at regular time points after gene therapy as a guide to the level of gene marking in CD3+ T cells, CD19+ B cells, CD56+ NK cells and CD15+ neutrophils (top to bottom). c, Change over time in the proportion of WASp-positive CD3+ T cells, CD19+ B cells and CD56+ NK cells after gene therapy.

Fig. 2. TCRβ repertoire analysis.

a, Shannon diversity of the TCRβ clonotypes analyzed in patients with WAS (P1, P2, P4–P8) at months 24 and 48 of post-gene therapy follow-up (left) and in four healthy donors (HD1–HD4) (right). b, The Spearman correlation between the TCRβ Shannon diversity at month 24 and the number of corrected CD34+ cells infused per kilogram, for each patient. The P value was calculated using the two-sided Spearman’s rank correlation test, and r is Spearman’s rank correlation coefficient. A regression line is represented in black and the 95% confidence interval is shown in gray. c, Circos plots showing V-gene and J-gene combinations in the repertoires analyzed in patients with WAS at month 48 of post-gene therapy follow-up (except for P8, shown at month 24) and in one healthy donor (HD3). The colored arcs define distinct Vβ and Jβ genes. The ribbon thickness is proportional to the frequency of a given combination.

Fig. 3. Restoration of T cell immune synapse assembly after gene therapy.

a, Representative confocal microscopy images of conjugates between the indicated T cells (T, upper cells) and monoclonal anti-CD3 antibody-coated P815 cells (P815, lower cells), after staining for F-actin, WASp and high-affinity LFA-1. The T cells from P5 were collected 55 months after gene therapy; comparable data were collected from additional patients before and after gene therapy (P2) or after gene therapy (P4 and P7). The data were reproduced in two independent experiments (including cell preparation, staining and image acquisition). b, Integrated fluorescence density for WASp in T cells forming conjugates with monoclonal anti-CD3 antibody-coated P815 cells in patients with WAS before and after gene therapy (43, 55, 48 and 41 months afterwards for P2, P5, P4 and P7, respectively), in HDs (HD5 and HD6), and in one non-treated patient with WAS (WAS 1). c, Integrated fluorescence density for high-affinity LFA-1 in the same cells as in b. Overall, 28–37 conjugates per sample were analyzed. Similar results were obtained in another independent experiment. Statistical significance was assessed by unpaired t-test (two-tailed) (***P < 0.0001); NS, not significant (P = 0.3866). Black horizontal lines in b and c represent the mean.

Fig. 4. B cell reconstitution after gene therapy.

a, Switched memory B cell subsets over time. b, IgG production in patients who discontinued intravenous immunoglobulin replacement therapy. Age-matched reference values are indicated by the gray lines (solid line, lower value; dashed line, upper value). c, WASp expression (mean fluorescence intensity (MFI) in arbitrary units) in switched memory, marginal zone and naive B cell subsets in patients (P) and healthy donors (HD7–HD11). d, The VCN in memory B cell (CD27+) and naive B cell (CD27−) subsets (n = 3 biologically independent samples). Data are presented as the mean ± s.e.m.

Fig. 5. Hemostasis and platelet profile after gene therapy.

a, Change over time in the platelet count for each patient after gene therapy. b, The Spearman correlation between platelet count and the level of gene marking (VCN) in CD15+ neutrophils after 3 years of follow-up for each patient (apart from P8, for whom the platelet count was corrected following splenectomy before gene therapy). The P value was calculated using the two-sided Spearman’s rank correlation test, and r is Spearman’s rank correlation coefficient. A regression line is represented in black and the 95% confidence interval is shown in gray. c, Platelet size, evaluated using flow cytometry. Each dot represents the mean size of washed platelets from healthy donors (HDs; n = 13), gene therapy-treated patients (P) and non-treated patients with WAS (WAS 2 and WAS 3). The results are expressed as the mean forward scatter height (FSC-H) ± s.d. and the dotted lines indicate the normal range (mean ± 2 s.d.). AU, arbitrary units. d, Platelet ultrastructure, which was analyzed once for each patient using transmission electron microscopy. e, The thrombin-induced aggregation of washed platelets was evaluated once for each patient (P) and healthy controls (HDs). f, WASp expression in patients was evaluated by western blotting. Dotted lines indicate that the samples were derived from the same gel but are non-contiguous. The graph shows the mean of WASp expression ± s.e.m. after normalization against CD41 expression from several independent experiments (HDs, n = 82; P4, WAS 2, WAS 3, n = 4; P5, n = 6; and P2, P7, n = 8). g, WASp expression was evaluated by immunofluorescence in platelets spread onto a fibrinogen matrix after activation with 20 µM ADP. The dot plot on the left shows the mean of the percentage ± s.e.m. of platelets expressing WASp, and the dot plot on the right shows the mean of the WASp expression (determined from the MFI per platelet) ± s.e.m. for five fields of view, which represent a total number of analyzed platelets of 816 for HDs, 170 for P2, 196 for P4, 175 for P5, 145 for P7, 158 for WAS 2 and 168 for WAS 3. In f and g statistical significance was determined using one-way analysis of variance with Dunnett’s post test for multiple comparisons. In f (right) and g (left), the exact P value was P < 0.0001 for all of the patients, and in g (right) the exact P value was P < 0.0001 for P2, P4, P5, WAS 2, WAS 3 and P = 0.0007 for P7. In c–g, all HDs were different donors, except in g (left and right) where HDs were the same.

Extended Data Fig. 1. Clinical and laboratory features of patients with WAS before and after GT.

Clinical and laboratory features of patients with WAS before and after GT: light pink: no disease; pink: mild disease; light red: moderate disease; red: severe disease. Eczema was graded with the SCORAD tool24. Infections and bleeding were graded in terms of recurrence and severity. Vasculitis was assessed according to the WAS severity scoring system4. Thrombocytopenia was defined as follows: grade 3 for a platelet count

Extended Data Fig. 2. Gene marking level.

Panel A, the VCN in the bone marrow (BM, white) subpopulations versus the peripheral blood (black) subpopulations in patients P2, P4 and P5; Panel B, VCN levels in the initial CD34 + drug product versus myeloid CD15 + cells 48 months after GT. Panel C, The Spearman correlation between the level of gene marking in CD15 + 48 months after GT and the number of corrected CD34 + cells infused per kilogram, for each patient. The pvalue (p) was calculated by using two-sided Spearman’s rank correlation test, r is Spearman’s rank correlation coefficient. A regression line is represented in black, the confidence interval in grey.

Extended Data Fig. 3. Immunological reconstitution after GT.

Immunological reconstitution after GT with age-matched reference values adjusted for each patient, CD3 + (Panel A), CD19 + (Panel B), CD56 + (Panel C) and CD15 + (Panel D). Age-matched reference values are indicated by the grey lines (mean values, dashed lines, lower and upper values, dotted lines).

Extended Data Fig. 4. Immunological reconstitution after GT.

Immunological reconstitution after GT of naive CD4 + T cells, CD4 + CD45RA + CD31 + cells (Panel A), naive CD8 + T cells, CD8 + CD45RA + CCR7 + cells (Panel B), Tcell receptor excision circles (sjTRECs) (Panel C) and immunoglobulin kappa-deleting recombination excision circles (sjKREC) (Panel D). Age-matched reference values are indicated by the grey lines (solid line: lower value; dashed line: upper value).

Extended Data Fig. 5. Immunoglobulin reconstitution after GT.

Immunoglobulin (Ig) reconstitution after GT. Panel A, IgM production over time; Panel B, IgA production over time. Age-matched reference values are indicated by the grey lines (solid line: lower value; dashed line: upper value).

Extended Data Fig. 6. Platelet analyses.

Panel A, Images show blood smears performed once for each GT-treated patients (P) or healthy donors (HDs) after May-Grünwald Giemsa staining. Arrows indicate the platelets. Panel B, TEM analysis of platelet ultrastructure in each patient (P). The box-and-whisker plots show the platelet shape defined as the ratio between the largest diameter and the smallest diameter derived from the TEM images performed once for each patient. Whiskers represent the upper and lower extreme values, the box correspond to the interquartile range, the center line is the median and the cross indicates the mean of 100 platelets for patients and 826 platelets for HDs. Statistical significance was determined in a one-way ANOVA with the Sidak post-test for multiple comparisons. The exact p value was p 

Extended Data Fig. 7. Ultrasound assessment of P2.

Ultrasound assessment of P2, showing three accessory spleens (measuring up to 3 cm in diameter) in the right hypochondrium.

Extended Data Fig. 8. Integration site (IS) analyses.

Panel A, The number of unique ISs detected in PBMCs at various points (in months) after gene therapy. Most of the samples were analyzed by sonic shearing in Illumina sequencing experiments (triangle), except that the early samples were analyzed using restriction endonucleases in 454 sequencing experiments (circle). Panel B, The Pielou diversity index for the IS population in PBMCs for the different patients. Panel C, A stacked bar representation showing the relative clone size (sonic abundance) of the TOP15 integration site clones detected over time for each patient.