Abstract
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.
Highlights
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency
We evaluated the ability of our gene editing protocol to restore functional WAS protein (WASp) expression in patient-derived primary WAS hematopoietic stem and progenitor cells (HSPCs) and their progeny
Peripheral blood (PB) or bone marrow (BM) HSPCs derived from four different WAS patients (Supplementary Fig. 2B) were gene targeted using the CRISPR/Cas[9] platform and each of the designed Associated Virus type 6 (AAV6) donor vectors
Summary
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. In terms of functional changes, there was a statistically significant increased level of phosphatidylserine exposure (Annexin V) and CD62P expression in WAS platelets compared to those derived from WT HSPCs upon activation; in both cases, gene editing using the W-pA, and to a minor extent the W-UTR, AAV6 donor construct restored normal WASp levels, while WW1.6 transduced cells exhibited partial correction of the defects (Fig. 3h, i).
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