WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation Muñoz Fernández, Pilar Tristán Manzano, María Sánchez Gilabert, Almudena Santilli, Giorgia Galy, Anne Thrasher, Adrian J. Martín Molina, Francisco This work was supported by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI12/01097, PI15/02015, and PI18/00337 (to F.M.). The CECEyU and CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069, and PAIDI-Bio326 (to F.M.) and PI-0407/2012 (to P.M). P.M. was supported by the European Union's Horizon 2020 through grant agreement no. 329284 (People Marie Curie Actions, Intra-European Fellowship [IEF], call: FP7-PEOPLE-2012-IEF) and by the Spanish ISCIII Health Research Fund through a postdoctoral fellowship "Sara Borrell" (CD09/00200). M.T.M. is funded by MCI through a fellowship FPU16/05467. A.G. is supported by funds from AFM-Telethon. We thank Dr. Claire Booth and Dr. Inmaculada Herrera for providing HDs and WAS patient blood samples and Ailsa Greppy for technical support with the animals (Western Labs, ICH-UCL). Some figures were created with BioRender.com. Supplemental Information can be found online at https://doi.org/10. 1016/j.omtm.2020.09.006. Transplant of gene-modified autologous hematopoietic progenitors cells has emerged as a new therapeutic approach for Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency with microthrombocytopenia and abnormal lymphoid and myeloid functions. Despite the clinical benefits obtained in ongoing clinical trials, platelet restoration is suboptimal. The incomplete restoration of platelets in these patients can be explained either by a low number of corrected cells or by insufficient or inadequate WASP expression during megakaryocyte differentiation and/or in platelets. We therefore used in vitro models to study the endogenous WASP expression pattern during megakaryocytic differentiation and compared it with the expression profiles achieved by different therapeutic lentiviral vectors (LVs) driving WAS cDNA through different regions of the WAS promoter. Our data showed that all WAS promoter-driven LVs mimic very closely the endogenous WAS expression kinetic during megakaryocytic differentiation. However, LVs harboring the full-length (1.6-kb) WAS-proximal promoter (WW1.6) or a combination of the WAS alternative and proximal promoters (named AW) had the best behavior. Finally, all WAS-driven LVs restored the WAS knockout (WASKO) mice phenotype and functional defects of hematopoietic stem and progenitor cells (HSPCs) from a WAS patient with similar efficiency. In summary, our data back up the use of WW1.6 and AW LVs as physiological gene transfer tools for WAS therapy. 2021-03-05T09:01:26Z 2021-03-05T09:01:26Z 2020-12-11 info:eu-repo/semantics/article Muñoz, P., Tristán-Manzano, M., Sánchez-Gilabert, A., Santilli, G., Galy, A., Thrasher, A. J., & Martin, F. (2020). WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation. Molecular Therapy-Methods & Clinical Development, 19, 220-235. [https://doi.org/10.1016/j.omtm.2020.09.006] http://hdl.handle.net/10481/66916 10.1016/j.omtm.2020.09.006 eng info:eu-repo/grantAgreement/EC/H2020/329284 http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess Atribución 3.0 España Elsevier