Discussion

Reporter gene assays are widely used to study the regulation of gene expression. However, this has previously only been possible using exogenous plasmid‐based overexpression technologies. We have developed a suite of endogenous reporter cell lines which measure natural levels of protein and promoter activity. Here we show their utility in functional genomics research and high throughput screening.

Using adeno‐associated virus based gene editing, a proprietary part of Horizon’s GENESISâ„¢ platform (which consists of rAAV, ZFN and CRISPR), we have engineered the endogenous HIF1A locus to generate an in‐frame NanoLuc® luciferase protein fusion in the HCT116 colorectal cancer cell
background. NanoLuc® luciferase is a small enzyme engineered for optimal performance as a luminescent reporter. The enzyme uses furimazine, a novel substrate, to produce a glow‐type luminescence that is approximately 150‐fold brighter than other luciferases. The bright nature of NanoLuc® luciferase enables us to detect activity at endogenous expression levels.

Validation experiments were first performed to assess the utility of the HIF1A NanoLuc® protein reporter in drug discovery and development processes. Protein turnover was detected by using cycloheximide and protein accumulation was measured using bortezomib to block protein
degradation. Both assays were multiplexed with CellTiter‐Blue® Cell Viability Assay ensuring effects were not due to changes in viability. Furthermore, it was demonstrated that a linear relationship between luciferase signal and cell number is maintained even at low cell numbers, and decay kinetics showed a stable signal half‐life, suggesting these assays would be amenable to highthroughput screening platforms.

A high‐throughput screen was then performed using the HIF1A NanoLuc® protein reporter. Cells were seeded into 384 or 1536 well plates and induction of HIF1A confirmed using 1% oxygen or the hypoxic‐mimetic CoCl2. Knockdown of Hif1A protein reporter signal was confirmed using YC‐1 and Topotecan, known HIF1A inhibitors. The high throughput screen was run in 1536‐well plate format using the NCGC Pharmaceutical Collection of approved and investigational drugs. The assay performed well with Z’ of 0.70 and CV of 6.1% and hit rate compared to viability is currently being interrogated. This assay is robust and suitable for a large scale library screening.

In conclusion, we have generated novel protein reporter cell lines by introducing NanoLuc®, allowing us to study, for the first time, the modulation of endogenous protein stability in the high‐throughput
setting. This technology could be applied to virtually any gene of interest and in a range of cell backgrounds, with resultant cell lines having applications in basic research as well as in high throughput compound library screens.

Jessica Hunt, Horizon Discovery