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

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.

Targeted Gene Delivery: Where to Land

“…Gene-specific strategy Limited to gene body mutations Urnov et al, 2005;Lombardo et al, 2007;Li et al, 2011;Genovese et al, 2014;Voit et al, 2014;Dever et al, 2016;Hubbard et al, 2016;Schiroli et al, 2017;Sweeney et al, 2017;Kuo et al, 2018;Wang et al, 2019Wang et al, , 2020aRai et al, 2020 (Hubbard et al, 2016;Kuo et al, 2018), and Wiskott-Aldrich Syndrome (Rai et al, 2020). Beside HSC and terminally differentiated blood cells, like B and T cells (Wang et al, 2016;Hung et al, 2018), AAV and nucleases have been the preferred method to achieve targeted transgene integration in many tissues in vivo (Suzuki et al, 2019;Kohama et al, 2020;Nishiguchi et al, 2020), especially the liver.…”

Cell‐Based Delivery Systems: Emerging Carriers for Immunotherapy

“…Genetic engineering techniques including gene editing, gene engineering, and gene reprogramming enable researchers to up‐regulate or down‐regulate specific genes, alter pathogenic genes or incorporate desired foreign genes, offering a powerful tool to customize the features, correct genetic defects, and control the fate of a variety of cells. [ 186 ] These can be achieved both by altering DNA to create inheritable changes, or using siRNA or mRNA to control RNA or protein expression while leaving DNA unchanged. [ 187 ] Genetic engineering provides technical support for many emerging cell therapies such as stem cell therapy and CAR‐T cell therapy.…”

Role of Thrombopoietin Receptor Agonists in Inherited Thrombocytopenia

“…However, a careful evaluation of the risk–benefit ratio must always be made [ 33 ]. Moreover, lentivirus-based gene therapy is already a proven therapy in WAS when there is not an optimal donor available for HSCT, although restoration of the platelet count may remain incomplete [ 43 , 44 ]. In the future, a risk profiling should be made case-by-case to identify candidates for gene therapy procedures, although currently this approach is not available for most forms of ITs, other than WAS [ 11 , 45 ].…”