905. The Endogenous WASp Promoter Fragments in SIN Lentiviral Vectors Drive Expression Preferentially in T Cells

Abstract

Top of pageAbstract Wiskott Aldrich Syndrome (WAS) is a human X-linked recessive immunodeficiency characterized by thrombocytopenia, eczema, aberrant T cell activation and cytoskeletal organization. Two regulatory regions regulate the WASp gene. The proximal promoter,5' and adjacent to the initiation site, contains binding sites for several transcription factors such as Sp1, Ets, c-Myb and PU.1. The distal promoter lies 6 kb upstream from the initiation site and is followed by an alternative first exon of the WASp gene. Expression of a transferred WASp cDNA using the WASp promoter may yield more effective correction than a constitutive viral promoter. More importantly, the WASp gene enhancer/promoter may be less likely to trans-activate cellular genes in genomic sequences flanking vector integration sites, than a strong retroviral LTR with ubiquitously-active enhancers. Thus, if the WASp promoter vector shows functional equivalency to the vector using the retroviral LTR enhancer/promoter, it may represent a safer construct for clinical applications. To test the feasibility of using the endogenous WASp promoter in SIN lentiviral (SMPU) vectors, a series of constructs were developed. Transcriptional control of eGFP was directed by either the retroviral enhancer/promoter of MND, the murine PGK gene promoter, the proximal WASp promoter, the distal WASp promoter or chimeras of the distal and proximal promoters. VSV-G pseudotyped viral supernatant was produced by transfection of 293 cells. Titre for all of the WASp promoter vectors was consistently 1–2 logs lower than MND or PGK vectors. Trandsduction of cell lines was performed with equal IU/ml resulting in no more than one proviral copy per cell. In all cells the highest level of expression (MFI) was observed from the MND LTR enhancer/promoter with the murine PGK and the WASp promoters at least 10-fold less bright. However, eGFP expression driven by the proximal WASp promoter results in preferential expression (>40%) in human T cell lines, although strict tissue specificity was not observed. The distal promoter was also active, but less than the proximal promoter. The chimeric promoters, with the distal and proximal fragments together in either order, had transcripts from both promoters, but transcripts from the proximal promoter were more abundant than from the distal promoter. Interestingly, the transcripts from the distal promoter were not of the expected size with transcription appearing to start at the 5' end of the fragment. Sequence analysis revealed a potential cryptic start site in the multicloning site at the 5' end of the distal fragment, however, mutation of this potential start site did not alter the distal promoter transcript. Analysis of the actual transcript start site is in progress. Vectors containing these promoters driving WASp expression have been constructed and shown by western analysis to express WASp. Transduction of murine WASp knockout bone marrow results in WASp expression 10 fold higher from the MND enhancer/promoter than from the proximal promoter. These constructs are being tested in the murine model of WASp and in human patient cell lines.