Targeting Gene Expression to Endothelial Cells in Transgenic Mice Using the Human Intercellular Adhesion Molecule 2 Promoter1

Abstract

Genetic engineering of donor animals in xenotransplantation research has been directed largely toward obtaining expression of various immunoregulatory molecules on vascular endothelium, the initial target of recipient antibody and complement. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. Using the promoter of the gene for intercellular adhesion molecule 2 (ICAM-2), which is constitutively expressed on all vascular endothelium, we have developed a system for endothelial cell gene targeting in vivo. A 334-basepair fragment from the 5' flanking region of the human ICAM-2 gene was used to drive the expression of human CD59 in transgenic mice. Strong and uniform expression of CD59 was observed on the endothelial cells of all blood vessels in the heart, kidney, lung, liver, and pancreas in the three lines of mice examined. Little or no expression was seen in other cell types, with the exception of neutrophils and monocytes. These results suggest that this small promoter region contains most of the signals necessary to endow it with endothelial cell specificity, making it a potentially valuable tool in areas ranging from xenotransplantation to gene therapy.

Tissue-specific gene therapy directed to tumor angiogenesis

“…These promoters include: von-Willebrand (vWF) factor that showed poor transfection efficiency, 14 intracellular adhesion molecule 2 (ICAM-2), that directed strong and specific expression in the EC of transgenic mice, 15 and the murine tie-2 promoter fragment that lacked EC specificity in vitro, but showed high and specific activity in transgenic mice. 16 However, these EC-spe-…”

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

“…The promoters for a number of genes with transcriptions restricted to endothelium have been sequenced and partially characterized; these include vascular cell adhesion molecule-1 (VCAM-1), 2 endothelial nitric oxide synthase (eNOS), 3 von Willebrand factor (vWF), 4 fms-like tyrosine kinase-1 (FLT-1), 5 tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE), 6 kinaselike domain receptor (KDR), 7 and intercellular adhesion molecule-2 (ICAM-2). 8 To date, no studies have directly compared candidate EC-specific promoters within the same viral gene-delivery system. Here, we document the gene expression profiles of adenoviral vectors using the candidate vWF, FLT-1, and ICAM-2 promoters in vitro, ex vivo, and in vivo.…”

Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity

“…Analysis of human promoters characterized for endothelial-specific expression led to the identification of fragments from the promoters of flt-1, 32 ICAM-2 34 and KDR 33 as suitable for transcriptional targeting. We have developed hybrid LTR retroviruses incorporating sequences from each of these and assessed them in vitro, on a cell line panel, and in vivo, in a subcutaneous xenograft model.…”

“…31 All have been subjected to some promoter characterization with regard to endothelial specificity, using transient transfection assays for flt-1 32 and KDR 33 or transgenic mice for ICAM-2. 34 Promoter fragments were amplified from genomic DNA such that cloning into the DEP backbone, immediately prior to the viral TATA box, would regenerate the original spacing relative to the TATA box or, for the ICAM-2 and KDR promoters lacking a TATA box, relative to the transcription start site (Figure 1c). In the latter cases, the LTR TATA box is expected to substitute for the initiator element found in such promoters, as has been previously seen for the murine terminal deoxynucleotidyl transferase promoter.…”