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.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.