Generation of a high titre retroviral vector for endothelial cell-specific gene expression in vivo.
Tumour growth is dependent upon a blood supply and is associated with the switch to the angiogenic phenotype. We are developing strategies for targeting gene expression to endothelial cells in the tumour vasculature. Recombinant retroviruses have been generated that incorporate regulatory sequences of the prepro-endothelin-1 (ppET1) promoter. Following reverse transcription and integration these modifications are duplicated in the proviral 5' LTR for transcription of the internal beta-galactosidase reporter gene. The titres and endothelial specificity of retroviral vectors harbouring different modifications have been analysed. In the optimal strategy, replacing the MLV enhancer with ppET1 promoter sequences containing the GATA and AP1 elements whilst maintaining sequences from the viral promoter resulted in endothelial cell-specific expression of the reporter gene, and viral titres comparable to those of the unmodified vector. A panel of endothelial and non-endothelial cells infected with the modified virus from a high titre producer clone showed a pattern of expression consistent with the activity of the endogenous ppET1 promoter. The modified LTR retained specificity in vivo, in subcutaneous tumours arising from the co-injection of tumour cells and irradiated virus producer cells. This simple model achieves high efficiency of transduction and can be used routinely for the screening of targeted retroviral vectors. Gene Therapy (2000) 7, 368-376.
Mavria
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Chester Beatty Laboratories, Institute of Cancer Research, 237, Fulham Road, London SW3 6JB, UK.
Jäger
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Porter
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D015139:Blotting, Southern; D005697:Galactosides / Q000378:metabolism; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007211:Indoles / Q000378:metabolism; D009368:Neoplasm Transplantation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012878:Skin Neoplasms / Q000235:genetics; D014407:Tumor Cells, Cultured; D014775:Virus Activation / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D005697:Galactosides; D007211:Indoles; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside