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.
Koldej and colleagues perform preclinical evaluation of three promoters in a self-inactivating lentiviral vector being developed for ex vivo gene therapy of Wiskott Aldrich Syndrome (WAS). They demonstrate that a gammaretroviral promoter drives robust green fluorescent protein expression in multiple hematopoietic lineages in a mouse bone marrow transplant model, as well as significant WAS protein expression in patient peripheral blood cells. They further demonstrate that inclusion of insulator sequences can effectively abrogate insertional activation of the LMO2 gene, which has previously been found to drive malignant transformation of retroviral vector–transduced cells in human trials.
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