Comparison of Insulators and Promoters for Expression of the Wiskott–Aldrich Syndrome Protein Using Lentiviral Vectors

Abstract

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.

Development of gene therapy for blood disorders: an update

“…49 We have also found that the g-retroviral promoter is much stronger than either the EF1a or the WAS 1.6-kb promoter in various model systems. 50 Several other studies suggested that a 1.6-kb promoter fragment from the WASp gene achieves adequate levels for correction in the preclinical model as well as normal levels of expression in human CD341 cells.…”

High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis

“…19,20 The MND(U3) region of a modified gammaretroviral LTR is a strong promoter/enhancer, and has been previously successfully used internally in an LV vector in a clinical trial of gene therapy for adrenoleukodystrophy, without evidence of genotoxicity after 10 years of follow-up; it has also been shown to adequately correct the WAS phenotype in preclinical studies. [33][34][35] To avoid high expression of a pore-forming protein in HSPCs, we restricted expression in HSPCs by using miR126 target elements. 25,26 Transgene expression in HSPCs in globoid leukodystrophy was found to be toxic to HSPCs, which was overcome by placing miR126 target sites in the vector.…”

Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector

“…In previous mouse gene therapy experiments, we found that the WS1.6 promoter did not effectively rescue WASp expression in all lineages including B cells and resulted in the acquisition of features of humoral autoimmunity 20 . In contrast, an SIN-LV using a synthetic promoter derived from a γ-retrovirus called MND (MPSV LTR, NCR deleted, dl587 PBS) 26 as an internal promoter rescued WASp expression in all affected lineages and reduced the risk of autoimmunity 20, 27, 28…”

“…Although strongly trans -activating promoters are associated with an enhanced risk of insertional transactivation, this risk can be diminished by including a chromatin insulator 28, 30, 31. Chromatin insulators act as boundary elements preventing interactions between adjacent chromatin domains 32, 33, 34.…”

“…HSCT with LV containing the 650-bp cHS4-insulated MND-GFP (650.MND.GFP) resulted in GFP expression in all hematopoietic lineages, including platelets, that was stable over time. The 650.MND.GFP LV also showed the highest GFP expression per viral integration 28 . Therefore, based on our combined efficacy and expression data, a producer cell clone capable of generating high-titer LV with the 650-bp cHS4 insulator and MND-WASp expression cassette (650.MND.hWASp) was developed for clinical use 27 .…”