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.