Engineered HSV-1 Vector Expressing IL-12

The pd27-IL12 shuttle plasmid was linearized by SwaI (NEB) digestion. E11 complementing cells that express Cas9 and sgRNAs targeting the native HSV genome^{76 (link)} were co-transfected with linear pd27B-IL12 and infectious d106S DNA. Infectious d106S DNA was isolated as described previously.^{42 (link)} The progeny viruses were harvested and fluorescence-negative plaques were isolated and purified three times. Each plaque isolate was analyzed for evidence of IL12 insertion and subsequent lack of GFP by PCR. Viral stocks of both d106S and d106S-IL12 were grown and titered on E11 complementing cells.^{43 (link)} B16^Nectin1 cells were infected at varying multiplicities of infection (MOIs) with d106S or d106S-IL12. Twenty-four hours following infection, the supernatant was harvested, and IL-12 production measured by IL12p40 ELISA (BioLegend).