Gene therapy for the treatment of Wiskott-Aldrich syndrome (WAS) presents an 
alternative to the current use of allogeneic bone marrow transplantation. We 
describe the development of a self-inactivating lentiviral vector containing 
chromatin insulators for treatment of WAS and compare a gammaretroviral (MND), 
human cellular (EF1α), and the human WASp gene promoter for expression patterns 
in vivo during murine hematopoiesis using the green fluorescent protein (GFP) 
marker. Compared with the EF1α and the WASp promoters, expression from the MND 
promoter in mouse transplant recipients was much higher in all lineages 
examined. Importantly, there was sustained expression in the platelets of 
secondary recipient animals, necessary to correct the thrombocytopenia defect in 
WAS patients. Analysis of WAS protein expression in transduced human 
EBV-immortalized B-cells and transduced patient peripheral blood mononuclear 
cells also demonstrated stronger expression per copy from the MND promoter 
compared with the other promoters. In addition, when analyzed in an LM02 
activation assay, the addition of an insulator to MND-promoter-containing 
constructs reduced transactivation of the LM02 gene. We propose a clinical trial 
design in which cytokine-mobilized, autologous, transduced CD34(+) cells are 
administered after myelosuppression.