Initial measurement of gene and signaling regulation

TNFR1/IL1R1 expression in hNPCs transduced with epigenome editing vectors was measured by qRT-PCR. Total RNA was harvested using Purelink RNA micro kit (Invitrogen). Complementary DNA (cDNA) synthesis was performed with purified RNA using the High-Capacity cDNA Reverse Transcription Kit with RNAse Inhibitor (Applied Biosystems). Subsequently, cDNA was used for quantitative PCR (qPCR) with Taqman assays (Applied Biosystems) for TNFR1 (Hs01042313_m1), IL1R1 (Hs00991002_m1), and GAPDH (Hs02758991_g1). TNFR1/IL1R1 expression was normalized to GAPDH expression, and analyzed as fold change in expression relative to the NTC group (ΔΔC_t method).

The NF-κB activity of epigenome-edited hNPCs treated with TNF-α/IL-1β was analyzed through the lentivirally introduced luminescent NF-κB reporter to determine whether our vectors are able to modulate functional signaling of TNFR1/IL1R1. One day before dosing, hNPCs were plated (6,000 cells/well) in 96-well plates in 100 μL of culture medium. They were then treated with a range (0, 0.15, 1, and 10 ng/mL) of TNF-α (TNFR1-edited and NTC cells) or IL-1β (IL1R1-edited and NTC cells).

After 24 h, NF-κB activity (firefly luciferase luminescence assay⁴⁵) and number of cells (CCK8 assay; Dojindo) were measured. Induction of NF-κB activity was normalized to number of cells and analyzed as a fold change in luminescence relative to each untreated control (0 ng/mL) group (NTC, TNFR1 edited, or IL1R1 edited).