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

Abstract

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 5Ј LTR for transcription of the internal ␤-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

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

“…25 Insertion into the DE backbone was also successful both in vitro and in vivo, while use of the DP backbone removed the specificity for PAE over the nonendothelial TE671 cells. 26 This is consistent with the elevated activity of DP in the latter.…”

“…Human cell lines of various origin were used, as well as a porcine aortic endothelial (PAE) cell line used in our previous studies as a positive control for endothelial cell activity. 25,26 Expression in 293 cells showed relatively little dependence on the presence of the viral enhancer (three-fold) compared to the other cell lines (10-to 20-fold). The 293 and PAE cells showed the most marked dependence on the proximal promoter (40-fold compared to 5-to 10-fold).…”

“…This model enables transduction of both tumour and stromal cells as the tumour is established and is useful for assessing changes to transcriptional control in vivo. 26 Irradiated producer cells continue to produce virus efficiently in vitro but are cleared in vivo by the time the tumours are established. For the control, b-galactosidase expression was widespread throughout the tumour but positive cells within the stroma with the morphology characteristic of endothelial cells were rare (Figure 5a).…”

“…24 This laboratory has previously adopted such a hybrid LTR strategy using fragments from the human preproendothelin-1 (ppET-1) promoter to direct endothelialspecific transcription. 25,26 Vector design considerations were to generate a single transcriptional unit in the target cell and to avoid the use of selectable markers that may modify performance and would be inappropriate for eventual clinical application. However, since there can be great heterogeneity in endothelial cells due to development, cellular context and proliferation, 27 it was not obvious that the choice of ppET-1 promoter was optimal.…”

A novel gene transfer strategy that combines promoter and transgene activities for improved tumor cell inhibition

“…Replacement of the enhancer region with heterologous transcription factor target sequences can alter both the level of viral expression as well as alter the spectrum of cells capable of expressing that virus. For example, replacement of the enhancer region with a polyoma virus enhancer (PyF101), 1,2 tetracycline-responsive elements (TRA99), 3 cytomegalovirus immediate-early enhancer (CMV-IE), 4 CD4 silencer, 5 or regulatory sequences from the prepro-endothelin-1 (ppET1) promoter 6 have resulted in altered viral expression patterns, including cell-type restricted expression (ppET1, CD4 silencer), repressible expression (TRA99) or expression in a wider range of cell types (PyF101, CMV-IE). The strategy of altering the LTR in order to change retroviral expression patterns is also effective for lentivirus-based vectors.…”