Lentiviral gene therapy corrects platelet phenotype and function in patients with Wiskott-Aldrich syndrome

Abstract

Background Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. Objective We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. Methods We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. Results We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. Conclusions Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome

“…The fundamental pathogenesis of platelet defects in WAS is still largely elusive, but both ineffective platelet production and their accelerated elimination in periphery have been implicated [26][27][28] . The former hypothesis has been confirmed by recent studies highlighting the presence of platelet-intrinsic defects that may accelerate their clearance 29,30 , such as a reduced size and granularity, dysregulated activation at steady state (high CD62P expression) and increased phosphatidylserine exposure which in turn may lead to increased platelet phagocytosis. To determine the ability of our gene editing strategy to correct the abnormalities observed in this cell lineage, modified WAS HSPCs were also differentiated into platelets and functional correction was tested at this stage.…”

“…P-values were calculated using oneway ANOVA with Bonferroni's comparison test (a, c, f-j myeloid cells has been linked to persistence of inflammatory complications and autoimmunity 12,13,43 , reliable and robust WASp restoration through a gene editing platform could potentially reduce such risks. Full reconstitution of WASp expression could also contribute to more reliable correction of thrombocytopenia, and therefore to application of this approach to patients with attenuated WAS who predominantly manifest thrombocytopenia and bleeding risk 12,13,29 . Although limited to an in vitro platelet differentiation system, our data suggest that targeted integration of the W-pA cassette can restore normal levels of WASp in megakaryocyte progenitors and mature platelets, correcting the platelet-intrinsic defects observed in WAS patients.…”

Gene therapy for Wiskott-Aldrich syndrome: History, new vectors, future directions

“…This may also be a result of improved platelet function and phenotype after treatment. 9 Autoimmunity improved after GT, 5,8 tolerance. However, in contrast to the results of other centers, 2 subjects treated in Boston with preexisting autoimmunity had no resolution after GT, in association with poor recovery of lymphocytes, including regulatory T cells.…”

Gene therapy for severe combined immunodeficiencies and beyond

“…This was enough to prevent severe bleeding but not to stop bleeding events completely. These results indicate that even after the transduction of multipotent HSCs and their differentiation into hematopoietic lineages (Scala et al, 2018), platelet production does not fully compensate for the destruction of WASp-deficient platelets (Sereni et al, 2019). It may well be that thrombocytopoiesis requires more progenitor cells than lymphopoiesis because of the high platelet turnover.…”