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. 2013 Nov 19;4:2773. doi: 10.1038/ncomms3773

Figure 1. Platelet-targeted lentiviral vector design.

Figure 1

(a) ITGA2B gene promoter fragments direct megakaryocyte-specific expression in luciferase reporter assay. Human pro-megakaryocytic, lymphocytic, erythrocytic and epithelial cell lines are transfected with a luciferase reporter construct under the control of one of three fragments of the human ITGA2B gene promoter beginning at either nucleotide −1218(green), −889(yellow) or −673(red) in-frame with the luciferase gene. A luciferase construct without a gene promoter served as a negative (−)control (peach), whereas a construct with a tissue-nonspecific gene promoter of the cytomegalovirus (CMV) is a positive (+)control (blue) arbitrarily assigned a value of 100% luciferase activity. Lysates of each cell line (x axis) were measured in duplicate on a luminometer to report the percent (%) luciferase activity (y axis) for each construct within each cell line. Cells were co-transfected with plasmid constructs encoding the marker gene, β-galactosidase, under the under the control of the CMV promoter to correct for variations in transfection efficiency. Shown is one representative of nine experiments where the error bars represent the s.d. from the mean value of measurements made in duplicate. (b) −889ITGA2B-BDDFVIII-WPTS lentiviral vector diagram. Shown is the region between the 5′-long terminal repeat (LTR) (left, blue box) and 3′-LTR without a U3 enhancer/promoter (right, blue box) to permit the ITGA2B promoter (yellow arrow) to direct megakaryocyte-specific synthesis of BDDFVIII (grey circle). The −889ITGA2B promoter binds GATA and Ets transcription factors for high-level gene expression in megakaryocytes and a repressor region inhibits gene expression in other lineages (yellow arrow). The central polypurine tract (cPPT, navy) and woodchuck hepatitis virus postregulatory element (WPRE, purple diamond) enhance efficiency of transgene expression. (c) −673ITGA2B-VWFSPD2-BDDFVIII-WPTS lentiviral vector diagram. The backbone of this vector is the same as described in b, except a smaller −673ITGA2B promoter (red arrow) was fused to DNA encoding the human VWF signal peptide (SP) and D2 domain (peach circle) to traffic BDDFVIII (grey circle) directly to platelet α–granules.