Generation of a high titre retroviral vector for endothelial cell-specificgene expression in vivo

Tumour growth is dependent upon a blood supply and is associated with the s witch to the angiogenic phenotype. We are developing strategies for targeti ng gene expression to endothelial cells in the tumour vasculature. Recombin ant retroviruses have been generated that incorporate regulatory sequences of the prepro-endothelin-1 (ppET1) promoter. Following reverse transcriptio n and integration these modifications are duplicated in the proviral 5' LTR for transcription of the internal p-galactosidase reporter gene. The titre s and endothelial specificity of retroviral vectors harbouring different mo difications 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 endotheli al cell-specific expression of the reporter gene, and viral titres comparab le to those of the unmodified vector A panel of endothelial and non-endothe lial cells infected with the modified virus from a high titre producer clon e showed a pattern of expression consistent with the activity of the endoge nous ppET1 promoter The modified LTR retained specificity in vivo, in subcu taneous tumours arising from the co-injection of tumour cells and irradiate d virus producer cells. This simple model achieves high efficiency of trans duction and can be used routinely for the screening of targeted retroviral vectors.