PB0153 - Evaluation of cell type-specificity of native enhancer elements from the F8 locus

Background: Remaining concerns with FVIII gene therapy include marked variability of factor levels and a decline in levels over time. These problems might be mitigated by the delivery of vectors possessing native regulatory elements to endothelial cells that normally express FVIII.

Aims: To investigate transcriptional potential of FVIII-Padua enhancer elements in liver and endothelial cells.

Methods: We investigated two regions from intron 1 of the F8 locus: Fragment C - 927 base pairs (bps) and a sub-region of this fragment (Fragment C’, 400 bps). These elements were cloned into a reporter plasmid in forward and inverted orientations. Constructs were transfected into HEK293T, HepG2, HUVEC and HLEC (lymphatic endothelial) cells. Luciferase expression was measured.

Results: In HEK293T, Fragment C’ and inverted C’ increased luciferase expression 36- and 21-fold (P < 0.0004 and 0.009, respectively). Forward orientation Fragment C increased luciferase expression just 5-fold, and inverted Fragment C resulted in ~1-fold increase. In HepG2 cells, Fragment C’ again resulted in marked enhancement of expression, increasing luciferase levels 52- and 47-fold for the forward and inverted orientation constructs, respectively (both P< 0.0001). Fragment C resulted in a small 2.5-fold increase in reporter gene expression. In HUVECs, Fragment C’ increased luciferase expression 17-fold (p=0.0001) and inverted C’ 13-fold (P=0.001). Fragment C resulted in 7- and 2-fold increases, respectively with forward and inverted insertion. Lastly, HLEC-derived expression was increased 27- and 31-fold with forward and inverted Fragment C’ (P=0.0016 and 0.0018), respectively. In these cells, Fragment C mediated 6- and 3-fold expression with forward and inverted constructs.

Conclusion(s): Significant increases in gene expression were observed in all cell types with the 400bp fragment in both orientations, consistent with the role of this sequence as a strong ubiquitous transcriptional enhancer. In contrast, minimal enhanced expression was observed with the 900bp fragment in all cells, suggesting presence of repressor element(s) in this sequence.