[Show abstract][Hide abstract] ABSTRACT: We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5' of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.
[Show abstract][Hide abstract] ABSTRACT: In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method of continually measuring expression of this marker gene after gene transduction. Standardly, its substrate, luciferin, is injected into the peritoneal cavity prior to bioimaging. Here we demonstrate that, compared with intra-peritoneal injection, intra-nasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed following administration of viral vectors based on adenovirus-5, AAV8 and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after non-viral PEI DNA delivery. Detection increased relative to the concentration of luciferin however a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intra-peritoneal injection, intra-nasal instillation yields around a ten-fold increase in sensitivity with an approximate thirty-fold reduction in the luciferin usage when bioimaging in the nasal and pulmonary airways of mice.
Human gene therapy 09/2008; DOI:10.1089/hgt.2008.023 · 3.76 Impact Factor