PROJECT SUMMARY – CORE 1 Nervous system tumors such as glioblastoma (GBM) remain amongst the most difficult to tumors treat. We have been interested in using oncolytic viruses, based on herpes simplex virus type 1 (HSV-1), as a selective nervous system tumor-killing virus. Considerable effort in the development of HSV-1 vectors has resulted in the engineering of oncolytic HSV vectors (oHSV), such as rQNestin34.51 that are non-toxic for normal cells, yet capable of selective replication in GBM tumor cells. To effectively use oHSV in animal tumor models it is necessary to propagate and purify oHSV to high titers. Our efforts in process development have resulted in scalable systems capable of manufacturing rQNestin34.5 and other oHSVs, and our protocols have been modified by Project 4 (Yu/Caligiuri) and the City of Hope Contract Manufacturing Facility to produce cGMP- grade oHSV-CCL5 for the upcoming Phase-I trial as part of the P01 Renewal. The primary goal of the oHSV Production Core will be to provide large quantities of research-grade concentrated and purified oHSV vectors for Project 1 (Glorioso; armed and unarmed rQNestin34.5v1 non-SYN and SYN), Project 2 (Chiocca; rQNestin34.5v1 and rQNestin34.5v2), Project 3 (Kaur; oHSV-rQ1 and oHSV-P10- aCD73mAb), and Project 4 (Caligiuri; oHSV-CCL5 and oHSV-αCD47-IgG1). Since the P01 started in 2012 we have produced, purified, and distributed over 60 oHSV stocks to the 4 projects while producing 23 MVBs with 36 oHSV stocks produced from 2017-2022. The oHSV Production Core will continue to work with the Projects to provide optimal vector quantity and purity while providing the support necessary to successfully exploit the available technology. We have shown that inclusion of dextran sulfate in production increased oHSV yield 15x, and that our stocks are devoid of contaminating exosomes. We have also engineered a cell line for efficient BAC removal, cloned a non-adherent cell line, determined that HEK293 cells are superior to Vero-based cells for optimal oHSV yield, and developed a novel assay to assess DNA genome containing oHSV particles. Should any of the engineered oHSV vectors tested in the Projects prove efficacious, the oHSV Production Core would provide support in transfer of SOPs and QA/QC assays to cGMP facilities for the production/purification technology for large-scale oHSV manufacture for possible Phase-I human clinical trials as seen with rQNestin34.5 and more recently with oHSV-CCL5 that underwent cGMP manufacturing by an outside vendor.