Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity.
Transcriptional targeting is an important aspect of developing gene therapy vectors in order to restrict transgene expression to selected target cells. One approach, when using retroviral vectors, is to replace viral transcriptional control elements within the long terminal repeat (LTR) with sequences imparting the desired specificity. We have developed such hybrid LTR retroviruses, incorporating sequences from each of the human promoters for flt-1, ICAM-2 and KDR, as part of our antivascular cancer gene therapy strategy targeting tumour endothelial cells. The chosen fragments were used to replace the enhancer or combined enhancer and proximal promoter regions of the viral LTR. All showed activity in primary human breast microvascular endothelial cells, with viruses incorporating ICAM-2 sequences exhibiting the greatest specificity versus nonendothelial cells in vitro and a marked alteration of specificity towards endothelial cells in a subcutaneous xenograft model in vivo. Moreover, our study documents the effect of enhancer and/or proximal promoter deletion on LTR activity and reports that differential dependence in different cell lines can give the false impression of specificity if experiments are not adequately controlled. This finding also has implications for other retroviral vector designs seeking to provide transcriptional specificity and for their safety with respect to prevention of gene activation at sites of proviral integration.
Institute of Cancer Research, Fulham Road, London, UK.
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Richardson
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Kaspers
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Porter
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000628:therapy; D011401:Promoter Regions, Genetic / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020079:Terminal Repeat Sequences / Q000235:genetics*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; C065289:ICAM2 protein, human