Dissecting enhancer/promoter function for high-level, persistent transgene expression

Gill DR, Pringle IA, Hyde SC, Oliveira CA

The European Society of Gene and Cell Therapy Conference, Florence, 2016

Persistent transgene expression is crucial for many gene therapy applications. We developed the hCEF enhancer/promoter comprising CpG-free versions of the human CMV enhancer (hC) and the human Elongation Factor 1a (EF) promoter, directing persistent luciferase expression in mouse lung (Hyde et al, 2008, Nat Biotech 26:549).

Persistent expression requires use of a CpG-free transgene cassette in plasmids and minicircles (Bazzani et al, 2016, Biomaterials 93:20), and is inflammation-free when all CpGs are removed. Deletion variants of the hC enhancer were generated to investigate the utility of hCEF. Complete deletion of the 302bp hC enhancer from hCEF resulted in 65-fold lower luciferase expression. When hC was replaced with the equivalent, 39% identical, murine enhancer (mC), high- level expression fell to background by day 7 (p<0.001). When the hC enhancer was progressively deleted, the distal 93bp was found to be both necessary and sufficient for persistent expression in the lung, although this expression profile was not recapitulated in the liver.

In silico analysis revealed five predicted transcription factor binding sites uniquely present in the minimal 93bp hC, but not the mC, enhancer, including SATB1.01 identified as a component of the nuclear matrix and highly expressed in mouse lung. Interestingly, expression from lentiviral vectors utilising hCEF was also higher and more persistent than conventional CMV and EF1a promoters in mouse nose and lungs (Alton et al, 2016 Thorax, In Press).

Together, these data support the exploitation of hCEF in both viral and non-viral vectors for lung gene therapy applications.