672. Lentiviral Vectors Targeting WASp Expression to Hematopoietic Cells, Efficiently Transduce CD34+ Cells and Correct Functions of Lymphocytes and Dendritic Cells from Wiskott- Aldrich Syndrome Patients

Abstract

Top of pageAbstract Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic cellular defects. To develop a safe and effective gene transfer system for this application, we designed SIN lentiviral vectors using 5' flanking sequences of the WAS gene as internal promoters to express a human WAS cDNA. This was shown to restrict the expression of the transgene to hematopoeitic cells, as is naturally the case for WAS. Further work was needed to validate this gene transfer system in multiple lineages of patients cells and to assess its performance in comparison with other lentiviral vectors utilizing strong internal promoters. Results showed that either short (0.5 kb) or long (1.6 kb) WAS promoters induced transgene expression in patients T, B and dendritic cells. Both of these promoter sequences behaved equivalently to each other but appeared to be transcriptionally weaker than the other promoters tested (PGK, EF1a, SFFV). Yet, they could correct the proliferation and IL-2 production defects in WAS T cells and the cytoskeletal anomalies in WAS DC, to the same level than achieved with stronger gene expression systems. They also induced gene expression in patient CD34+ cells without apparent hematopoietic toxicity. Thus, several robust SIN lentiviral expression systems exist to express the human WAS transgene. None of those tested here led to transgene over-expression or hematopoietic toxicity and all achieved functional correction in several lineages of patients cells. Having the therapeutic transgene under control of an endogenous WAS promoter sequence appears to be an effective and safe strategy. This preferentially targets the therapeutic transgene to the hematopoietic system, and may allow its physiological regulation in various lineages of cells. The lower strength of the WAS promoters may also reduce the risk of trans-activating neighbouring genes upon integration of the cassette. These results support further development of such lentiviral vectors for clinical application in WAS.