Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

Abstract

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott-Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.

Wiskott-Aldrich syndrome protein-deficient hematopoietic cells can be efficiently mobilized by granulocyte colony-stimulating factor

“…Indeed, the background of the mice determined the efficiency of hematologic recovery and this was particularly visible with B-cell recovery following transplantations. As previously described, 28 the lack of WASp caused an intrinsic defect in B-cell recovery which can be restored upon transplantation of WT progenitor/stem cells but not to the full extent in WKO recipient mice. Intrinsic B-cell defects in WASp deficiency haematologica | 2013; 98(8) Figure 4.…”

“…Thus, WASp deficiency causes an intrinsic defect in B lymphoid recovery confirming, in part, our own prior results. 28 In contrast to the lymphoid compartment, we observed a clear impact of mobilization on granulocyte recovery after transplantation. The transplantation of mobilized peripheral blood cells from WKO mice provided higher counts of granulocytes in the blood and spleen compared to transplantation of bone marrow, although this was not seen in the bone marrow compartment ( Figure 3D,E,F).…”

X-linked thrombocytopenia (XLT) due to WAS mutations: clinical characteristics, long-term outcome, and treatment options

“…10,[22][23][24] Hematopoietic stem cell gene therapy might in the future offer an alternative approach in patients lacking a suitable donor. [25][26][27] Generally accepted treatment policies do not exist for patients exhibiting the XLT phenotype, in whom HSCT would seem like an excessively risky procedure if they have thrombocytopenia and eczema only. Although it has been assumed that patients with XLT have a lower risk of cancer or autoimmunity than patients with WAS, this has never been formally examined.…”

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

“…Vector infectivity is calculated as the ratio between titer and particles. 50 Titers obtained ranged from 5.8E8 transducing units (TU)/mL to 1.1E9 TU/mL. LV_int2.1, LV_int2.6 and LV_ex2.1 were used at MOI 20 together with 8 mg/mL of polybrene in exon 2 duplicated myoblasts cell lines.…”