WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation

Abstract

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.

Physiological lentiviral vectors for the generation of improved CAR-T cells

“…We therefore focus on the WAS gene promoter because WASP is only expressed in hematopoietic cells, is involved in the formation of the immunological synapse, and acts as an adaptor of TCR signals. 17,18 We have previously developed different WAS-gene-based promoters and identified the AW promoter, harboring fragments from the alternative and proximal promoter, as the best option to express transgenes in hematopoietic cells, [19][20][21] which makes it an attractive candidate for T cells expression. Here, we showed that, indeed, WAS-promoterdriven LVs (AW-LVs) partially mimicked the TCR (CD3)-expression kinetic, with a small down-regulation upon stimulation and recovered basal levels in 5-7 days.…”

“…Since the TRAC promoter in human mature T cells is not well characterized, we focus on a well-defined promoter that controls the expression of the WAS protein (WASP), which is involved in the formation of the immunological synapse and in translating TCR signals to several T cell functions. 17,18 We reason that WAS-promoter-driven LVs [19][20][21] could be an interesting option to express CARs due to their moderate expression levels and the functional relationship with the TCR. Here, we showed that, indeed, WAS-promoter-driven LVs partially mimicked the TCR expression kinetic both in EGFP-and CAR-expressing LVs, with a small down-regulation upon stimulation and recovering basal levels (prior stimulation) in 5-7 days.…”

Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells

“…We therefore focus on the WAS gene promoter because WASP protein is only expressed in hematopoietic cells, is involved in the formation of the immunological synapse and acts as an adaptor of TCR signals 14,15 . We have previously developed different WAS -gene based promoters and identified the AW promoter, harbouring fragments from the alternative and proximal promoter, as the best option to express transgenes in hematopoietic cells 8,11,16 . Here we showed that, indeed, WAS -promoter driven LVs (AW-LVs) partially mimicked the TCR expression kinetic, with a small downregulation upon stimulation and recovering basal levels in 5-7 days.…”

“…Since the TRAC promoter in human mature T cells is not well defined, we focus in a well-defined promoter that controls the expression of the WASP protein, which is involved in the formation of the immunological synapse and in translating TCR signals to several T cells functions 14,15 . We reason that WAS- promoter driven LVs 8 11 16 could be an interesting option to express CARs due to their moderate expression levels and the functional relationship with the TCR. Here we showed that, indeed, WAS -promoter driven LVs partially mimicked the TCR expression kinetic both in eGFP- and CAR-expressing LVs, with a small down-regulation upon stimulation and recovering basal levels in 5-7 days.…”

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 ].…”