Project Summary/Abstract Genetically engineered neural stem cells (NSCs) are a promising therapy for the highly aggressive brain cancer Glioblastoma (GBM). Engineered NSCs have unique tumor-homing capacity that allows them to deliver anti-cancer gene products directly into local and invasive GBM foci. Preclinical studies by our group and others have shown tumoricidal NSCs routinely reduce orthotopic GBM xenografts between 70-90% and significantly extend survival of tumor-bearing mice. Yet, these dramatic initial reductions in GBM volumes are not maintained and treatment durability remains a major challenge for NSC-based therapy. GBM escape occurs after treatment with NSCs carrying different therapeutic payloads and in pre-clinical models of both solid and post-surgical GBM. We recently discovered that novel tumor-homing drug delivery vehicles with robust anti- cancer activity can be developed from ?induced neural stem cells? (iNSCs) using cellular reprogramming technology, referred to as transdifferentiation (TD). Tumoricidal iNSC therapy reduced GBM xenografts 230- fold in 4 weeks and more than doubled survival. Similar to wild-type NSC therapy, the tumors were not eradicated and the GBMs re-developed. The events mediating the regrowth of GBMs in response to single- agent NSC/iNSC therapy are unknown. Our results show that transplanted iNSCs drug carriers are cleared from the brain, but repeated intracerebroventricular (ICV) infusion restores carrier levels. We also have evidence that GBM cells become resistant to iNSC-delivered drugs. This allows us to hypothesize that GBM resistance to iNSC therapy can be overcome by repeat administration to address carrier loss and multi-agent iNSC delivery to address tumor resistance. With this grant we propose to test this hypothesis, defining the events that contribute to the dynamic adaption of GBM during NSC treatment and develop strategies to convert the initial tumor kill into sustained GBM suppression. We will investigate carrier clearance, homing, and tumor resistance throughout GBM adaption and recurrence. We will then modulate iNSC therapy through repeated dosing via ICV infusion and delivery of iNSCs carrying multi-drug payloads with the goal of improving treatment durability by overcoming iNSC loss and the emergence of GBM foci that are resistant to single-agent treatments. All testing will be done using our novel surgical resection models of murine-derived GBM cells in immune-competent animals and patient-derived CD133+ human GBM cells to maximize the clinical relevancy of our finding and understand the impact of the immune system on iNSC treatment durability. The results of these studies are essential for creating durable NSC-based tumor therapies capable of producing long-lasting GBM suppression in patient trials.